JP6998057B2 - Nerve injury treatment transplant material containing dental pulp cells - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Oral presentation at the 60th Annual Meeting of the Medical Biology Research Society of the Brain on January 23, 2016.

Application of Article 30, Paragraph 2 of the Patent Law Published in the lecture abstracts of the 20th Industry-Academia Collaboration Forum sponsored by the Dental Regenerative Medicine Industry-Academia Collaboration Conference issued on February 17, 2016. In addition, on March 17, 2016, an oral presentation was given at the lecture of the 20th Industry-Academia Collaboration Forum hosted by the Dental Regenerative Medicine Industry-Academia Collaboration Conference.

Application of Article 30, Paragraph 2 of the Patent Law Published in the abstracts of the 15th Annual Meeting of the Japanese Society for Regenerative Medicine issued on March 7, 2016. In addition, an oral presentation was made at the 15th Annual Meeting of the Japanese Society of Regenerative Medicine on March 17, 2016.

Application of Article 30, Paragraph 2 of the Patent Law Published in the abstracts of the 70th Annual Meeting of the Japanese Oral Science Society issued in April 2016. In addition, on April 17, 2016, an oral presentation was made at the 70th Annual Meeting of the Japanese Oral Science Society.

Application of Article 30, Paragraph 2 of the Patent Act Published in the IADR PBRG Symposium 2016 conference presentation abstracts issued in June 2016. In addition, an oral presentation was made at the academic meeting of IADR PBRG Symposium 2016 on June 27, 2016.

The present invention relates to a transplant material for treating nerve injury containing dental pulp cells and a method for producing the same.

The spinal cord is a neurotransmitter pathway between the brain and the periphery, and when it is damaged, it causes serious physical disabilities such as motor paralysis and sensory disturbance. Restoration of physical function lost due to spinal cord injury is extremely rare, and patients are severely restricted in their subsequent lives. In Japan, there are about 100,000 patients with SCI, and about 5,000 are newly injured each year. Although the development of radical treatments has become a social urgent need, none of the prior arts currently advancing in human clinical research have shown clear therapeutic effects.

In recent years, it has been reported that transplantation of human dental pulp cells remarkably recovers motor disorders in spinal cord injury model animals. Here, cell transplantation therapy can be divided into autologous transplantation and allogeneic transplantation. Autologous transplantation is a method of selecting and increasing cells of the patient's own tissue or cells derived from the autologous tissue, or inducing differentiation and transplanting. As typical examples of autologous transplantation, autologous tooth transplantation, autologous skin grafting, etc. are actually performed in clinical practice. In the case of autologous transplantation therapy, immune rejection can be avoided, but there are problems of invasion associated with excision of transplanted tissue or tissue-derived cells, individual differences in transplantation effect, and time and cost required for cell preparation.

On the other hand, allogeneic transplantation is a method of transplanting tissue or tissue-derived cells of another person. Immune rejection must be considered when using the cells of others. The immune response, which is a self-defense function, conversely hinders transplantation treatment. In recent years, advances in immunosuppressive agents have made it possible to suppress rejection, and organ transplantation and hematopoietic stem cell transplantation have made great strides. However, even with the current improved immunosuppressants, immunorejection occurs at a rate of about 20%, and there are some cases where treatment of immunorejection is extremely difficult, so safety concerns remain.

When considering allogeneic transplantation, dental pulp cells have several advantages as a source of transplanted cells. One can be collected from extracted teeth such as deciduous teeth and intellectual teeth that are originally treated as medical waste (10 million permanent teeth are discarded annually), and induction and culture methods have been established (non-patent). Since Patent Document 1), it can be used as an abundant source of transplanted cells. Here, immune rejection is based on attacking cells that are judged to be non-self by collating HLA antigens, but using HLA haplotype homodonor cells halves the types of antigens, increasing the number of patients who can adapt.

According to a recent report, it is estimated that if about 50 lines of HLA haplotype homozygous cells are prepared, about 80% of Japanese can be covered (Non-Patent Document 2). In other words, if dental pulp cells with abundant sources can be used for treatment, cells of HLA haplotype homodonors with less immune rejection can be collected, and the strategy using this as a transplantation source becomes realistic.
The present inventors have previously shown that treatment of dental pulp cells isolated from dental pulp tissue with FGF2 and transplantation can enhance the therapeutic effect of cell transplantation on spinal cord injury (for example, Patent Documents). 1). In addition, Patent Document 1 is a method for producing a transplant material for treating nerve injury, which comprises culturing dental pulp stem cells used in the transplant material in a medium substantially free of growth factors other than FGF2. Is disclosed.

WO2014 / 185470

Liu et al., Methods Enzymol, 2006, 419: 99-113 Okita et al, Nat Methods, 2011, 8: 409-412

So far, methods for isolating dental pulp cells from teeth and dental pulp tissues have been investigated, and establishment of a method for culturing dental pulp cells suitable for regenerative medicine applications has been investigated. Here, when the dental pulp cells are used for regenerative medicine, it is necessary to understand the difference in the cell properties of each donor from which the dental pulp cells are derived, as an important issue apart from the isolation method and the culture method. .. Not all dental pulp cells have a nerve damage recovery function because cells vary from person to person not only by age and gender but also by gene expression pattern. That is, depending on the donor from which it is derived, for example, the presence of dental pulp cells that do not have a therapeutic effect on nerve damage is assumed. In support of this, the present inventors have clarified that there is a remarkable difference in the effect of restoring motor function in a model animal after cell transplantation due to the difference in the donor of dental pulp cells (for example, Patent Document 1). .. In this way, in order to realize allogeneic transplantation therapy of dental pulp cells, it is necessary to clarify the difference in the properties of dental pulp cells among donors that correlates with the clinical effect, and to narrow down the biomarkers that can be examined before transplantation. Is an important issue.

However, there have been no reports on the differences in cell properties due to differences between donors and the properties of dental pulp cells that are preferable for the treatment of nerve damage. In particular, changes in the properties of dental pulp cells due to treatment with FGF2 and differences in the properties of dental pulp cells due to differences in donors were unknown. Therefore, the present invention identifies the properties that the FGF2-treated dental pulp cells should have, which are dental pulp cells used as a transplant material for treating nerve damage, and provides a transplant material for treating nerve damage containing the dental pulp cells. The challenge is to provide.

The present inventors have found that by treating dental pulp cells isolated from dental pulp tissue with FGF2, dental pulp cells preferable as a transplant material for treating nerve injury can be obtained. Here, the dental pulp cells treated with FGF2 express a marker of immature nerve cells after transplantation to the site of spinal cord injury, as compared with dental pulp cells cultured with MSCGM, which is a medium for stem cell culture without FGF2 addition. It has been confirmed that the cells have differentiated into cells (Non-Patent Document 1).
It should be noted that such a property supports that the dental pulp cells treated with FGF2 are useful for the treatment of nerve damage.

However, as described above, it has been clarified that even dental pulp cells treated with FGF2 do not have a therapeutic effect on nerve injury due to differences in donors when transplanted into spinal cord injury model animals. As a result of further diligent studies, the present inventions have found the properties of FGF2-treated dental pulp cells, which are particularly important for the treatment of nerve damage. The present invention has been completed based on this finding.
In addition, the present inventors show the results of comprehensive analysis and comparison of gene expression of dental pulp stem cells derived from different donors in dental pulp stem cells cultured with MSCGM without FGF2 in Patent Document 1. However, Patent Document 1 does not report analysis and comparison of changes in the properties of dental pulp cells due to FGF2 treatment, and analyzes and compares the effects of FGF2 treatment among dental pulp cells derived from donors having different therapeutic effects. It's not a thing. Therefore, in the FGF2-treated dental pulp cells to be used as a therapeutic implant for nerve injury, the properties that are indicators of the therapeutic effect on nerve injury have not been specified.

That is, the present invention is as follows:
The present invention is, as one aspect,
[1] The present invention relates to a transplant material for treating nerve injury, which comprises dental pulp cells in which the GABRB1 gene is expressed and has resistance to active oxygen.
Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[2] The transplant material for treating nerve damage according to the above [1].
The dental pulp cells are characterized by being treated with FGF2.
Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[3] The transplant material for treating nerve damage according to the above [2].
The FGF2-treated dental pulp cells are characterized by having an increased expression level of the GABRB1 gene as compared with the dental pulp cells not treated with FGF2-.

Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[4] The transplant material for treating nerve damage according to any one of the above [1] to [3].
The dental pulp cells are characterized by further expressing at least one gene selected from the group consisting of the MMP1 gene, the DRD2 gene, the ABCA6 gene, the TMEM100 gene, the THBD gene, the NTSR1 gene, and the SCG2 gene.
Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[5] The transplant material for treating nerve damage according to any one of the above [1] to [4].
The FGF2 treatment is characterized in that it is a culture using a medium containing FGF2 at a concentration of 5 ng / ml or more.
Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[6] The transplant material for treating nerve damage according to the above [5].
The culture using the medium containing FGF2 is characterized by being carried out for at least 6 days.

Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[7] The transplant material for treating nerve damage according to the above [3].
Compared with the dental pulp cells not treated with FGF2, the increased expression level of the GABRB1 gene is 10 times or more.
Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[8] The therapeutic material for transplantation of nerve injury according to any one of the above [1] to [7].
The nerve injury is characterized by spinal cord injury, cerebral infarction, intracerebral hemorrhage, submucosal hemorrhage, spinal cord hemorrhage, nerve compression injury due to herniated disk, sciatica, or peripheral nerve injury due to diabetes.
Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[9] The therapeutic transplant material according to any one of the above [1] to [8], which is used in combination with an active oxygen scavenger for the treatment of nerve injury.
Further, the transplant material for treating nerve injury of the present invention is, in one embodiment,.
[10] The transplant material for treating nerve damage according to the above [9].
The active oxygen scavenger is characterized by being at least one active oxygen scavenger selected from the group consisting of edaravone, vitamin C, Nrf2 inducer, and glutathione activity inducer.

Further, the present invention is in another aspect.
[11] A method for treating nerve damage, which is a method for treating nerve damage.
The present invention relates to a therapeutic method including a step of transplanting the transplant material for treating nerve injury according to any one of the above [1] to [8] to a nerve injury site.
Moreover, the method for treating a nerve injury of the present invention, in one embodiment,
[12] The present invention relates to a therapeutic method comprising a step of transplanting a therapeutic implant for nerve injury according to i) or ii) to a nerve injury site in combination with an active oxygen scavenger.
i) The therapeutic implant for nerve injury according to any one of claims 1 to 8, or
ii) A transplant material for the treatment of nerve damage containing dental pulp cells in which the expression level of the GABRB1 gene is increased by FGF2 treatment.

Further, the present invention is in another aspect.
[13] A method for producing a transplant material for treating nerve injury.
The process of measuring the expression of the GABRB1 gene in dental pulp cells
The present invention relates to a production method including a step of selecting dental pulp cells expressing the GABRB1 gene.
Further, the method for producing a therapeutic implant for nerve injury according to the present invention is, in one embodiment,.
[14] The method for producing a therapeutic implant for nerve injury according to the above [13].
At least one gene selected from the group consisting of MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene is further measured, and at least one gene selected from the group is expressed. It is characterized by further including a step of selecting a dentin cell.
Further, the method for producing a therapeutic implant for nerve injury according to the present invention is, in one embodiment,.
[15] The method for producing a therapeutic implant for nerve injury according to the above [13] or [14].
It is characterized by comprising culturing the dental pulp cells in a medium containing FGF2 prior to the step of measuring the gene expression of the dental pulp cells.

Further, the method for producing a therapeutic implant for nerve injury according to the present invention is, in one embodiment,.
[16] The method for producing a therapeutic implant for nerve injury according to any one of the above [13] to [15].
The culture in the step of culturing the dental pulp cells in a medium containing FGF2 is characterized by being carried out for at least 6 days.
Further, the method for producing a therapeutic implant for nerve injury according to the present invention is, in one embodiment,.
[17] The method for producing a therapeutic implant for nerve injury according to any one of the above [13] to [16].
The step of selecting dental pulp cells expressing the GABRB1 gene is a step of selecting dental pulp cells having a GABRB1 gene expression level that is 10 times or more higher than the GABRB1 gene expression level of FGF2-untreated dental pulp cells. It is characterized by being.
Further, the method for producing a therapeutic implant for nerve injury according to the present invention is, in one embodiment,.
[18] The method for producing a therapeutic implant for nerve injury according to any one of the above [13] to [17].
The nerve injury is characterized by spinal cord injury, cerebral infarction, intracerebral hemorrhage, submucosal hemorrhage, spinal cord hemorrhage, nerve compression injury due to herniated disk, sciatica, or peripheral nerve injury due to diabetes.

According to the therapeutic transplant material for spinal cord injury, which comprises the dental pulp stem cells expressing the GABRB1 gene and having active oxygen resistance according to the present invention, it has excellent cell engraftment after cell transplantation and has excellent cell engraftment property. Has an excellent therapeutic effect on spinal cord injury.

The evaluation criteria of the BBB score are shown. FIG. 2a shows transplantation of DP-1 strain dental pulp cells or DP31 strain dental pulp cells (DP1-FGF2 (-) or DP31-FGF2 (-)) untreated with FGF2 into a spinal cord injury model rat. Alternatively, the results of open field motor function evaluation based on the BBB score after administration of PBS are shown. Error bars indicate standard deviation (± S.D.). The significance test was performed by the Bonferroni post hoc test after the two-way ANOVA. FIG. 2b is a graph showing the results of measuring the electrophysiological action potential through the completely cut T10 site at the time of electrical stimulation of 0.6 mA. FIG. 3a shows the results of open field motor function evaluation based on the BBB score after administration of FGF2 or PBS to a spinal cord injury model rat. Error bars indicate standard deviation (± S.D.). The significance test was performed by the Bonferroni post hoc test after the two-way ANOVA. Asterisks show statistical significance compared to control groups treated with PBS (* P <0.05, ** P <0.01). FIG. 3b is a graph showing the results of measuring the electrophysiological action potential via the T10 site, which is the injured site, when FGF2 was administered to the spinal cord injury model rat and the electrical stimulation was performed at 0.6 mA. .. Only one of the four animals treated with FGF2 was able to detect action potentials beyond the injured site. FIG. 4a shows human dental pulp cells from different donors treated with FGF2 (DP1-FGF2 (+), DP31-FGF2 (+), DP165-FGF2 (+), DP296-FGF2 (+), PBS administration (control)). The results of open field motor function evaluation by BBB score when transplanted into a spinal cord injury model rat are shown. Error bars indicate standard deviation (± S.D.). The significance test was performed by the Bonferroni post hoc test after the two-way ANOVA. An asterisk (*) indicates that it is statistically significant compared to the control group to which PBS was administered (* P <0.05, ** P <0.01, *** P <0.001). FIG. 4b is a graph showing the results of transplanting human dental pulp cells derived from each donor into a spinal cord injury model rat and measuring the electrophysiological action potential via the T10 site, which is the damaged site, when 0.6 mA electrical stimulation was applied. Is. FIG. 4c shows a graph plotting the latency of each section in FIG. 4b. The significance test was performed by Tukey's multiple comparisons test after two-way ANOVA. The asterisk indicates that it is statistically significant compared to all plots (** P <0.01). The error bar indicates the standard error (± SEM) (n = 3). FIG. 5a shows an image of a state in which 4 × 10 ⁶ human dental pulp cells treated with FGF2 were transplanted into the vitreous cavity of a rat eye and 3 weeks after the transplantation. FIGS. 5b and 5C also show a cut surface image of the vitreous cavity taken out 3 weeks after transplantation. The scale bar indicates 500 μm. FIG. 6 shows an immunohistochemically stained image of the sagittal plane of the spinal cord 8 weeks after transplantation of DP165-FGF2 (+) or DP296-FGF2 (+) into a spinal cord injury model rat. 6a-c show images of the DP165-FGF2 (+) transplanted area, and FIGS. 6d-f show images of the DP296-FGF2 (+) transplanted area. In the center of the image of FIG. 6a, a GAP-43 positive growth cone was detected among the GFAP positive cells. FIG. 6b shows a high magnification image of the area shown by 1 in FIG. 6a. FIG. 6c shows a high magnification image of the caudal ventral area of the DP296-FGF2 (+) transplanted plot. Myelinated regenerated axons could be observed from the center to the ventral caudal side. In FIG. 6d, almost no GAP43-positive cells were observed. FIG. 6e shows a high magnification image of the area shown by 3 in FIG. 6d. FIG. 6f shows a high magnification image of the caudal ventral area of the DP296-FGF2 (+) transplanted plot. In FIG. 6f, the myelinated axons had disappeared. The scale bar in FIG. 6 shows 500 μm in FIGS. 6a and 6d, and 100 μm in FIGS. 6b, 6c, 6e, and 6f. The arrowheads in FIGS. 6a and 6d indicate the center of the injured site. 7a-c show fluorescence-stained images of the spinal cord at 8 weeks in the PBS-administered group (FIG. 7a), DP165 transplant group (FIG. 7b), and DP296 transplant group (FIG. 7c) after spinal cord injury. The scale bar indicates 1.0 mm. FIG. 8 is a graph showing the expression level of GABRB1 in a group treated with FGF2 or a group not treated with FGF2 for human dental pulp cells derived from each donor. The error bars indicate the standard deviation (± S.D.) (n = 3). An asterisk (*) indicates statistically significant compared to the FGF2 untreated group in each cell line (* P <0.05, ** P <0.01). FIG. 9 shows a graph of the results of real-time PCR quantifying the expression of specific genes in the FGF2-treated group or the FGF2 non-treated group for human dental pulp cells derived from each donor. The error bars indicate the standard deviation (± S.D.) (n = 3). An asterisk (*) indicates that it is statistically significant compared to the FGF2 untreated group (* P <0.05 and *** P <0.001). FIG. 10 shows the following Example 3. It is a schematic diagram which shows the culture schedule of each human dental pulp cell used for "the effect of FGF2 treatment on the resistance of human dental pulp cells to active oxygen". FIG. 11 is a graph showing the results of an active oxygen resistance test (MTT assay) on human dental pulp cells cultured in each culture schedule shown in FIG. FIG. 12 shows the culture method of the S / F group and the F / S group in each culture schedule shown in FIG. 10, and is an active oxygen tolerance test of human dental pulp cells when the number of culture days is further changed (FIG. 12). It is a graph which shows the result of MTT assay). FIG. 13 shows a fluorescence microscopic image of a longitudinal cross section of the spinal cord in a combination group of FGF2-untreated DP31 strain human dental pulp cells (DP31-S) transplanted with edaravone administration (daily). 13B and 13C are enlargements of the region shown in FIG. 13A. The scale bars in FIGS. 13A and 13C show 1 mm and 200 μm, respectively. The right and left sides of the image show the caudal and rostral sides of the spinal cord, respectively. FIG. 14 (A) shows a spinal cord injury site of a spinal cord injury model rat transplanted with DP31-S, and shows an image (20 magnification) showing the state 7 weeks after the cell transplantation. FIG. 14 (B) shows an image (20 magnification) showing a spinal cord injury site of a spinal cord injury model rat transplanted with DP31-S and combined with edaravone administration, and showing the state 7 weeks after the cell transplantation. .. FIG. 14 (C) shows the number of GFP-positive cells (mean) at the spinal cord injury site 7 weeks after the day of transplantation into the spinal cord injury model rat in the DP31-S transplantation group and the group of DP31-S transplantation and edaravone combination. It is a graph which shows the value). The values are shown as mean ± standard error. The significance test was performed by Student's t-test (* p <0.05). The scale bar indicates 200 μm. FIG. 15 shows a group (n = 5) in which dental pulp cells (DP31-S) were transplanted and edaravone was administered in combination with a spinal cord injury model rat, and a group (n = 9) in which dental pulp cells (DP31-S) were transplanted. ), And is a graph showing the time course of the BBB score in the group (n = 3) to which edaravone was administered daily. The values are shown as mean ± standard error. Significant differences were tested by Tukey's multiple comparisons test after two-way ANOVA (asterisks (*) indicate comparisons with control groups: * p <0.05, ** p <0.01, ** * p <0.0001, **** p <0.0001. † indicates a comparison with the dental pulp cell transplantation group: †† p <0.01, ††† p <0.001).

The present inventions have properties of FGF2-treated dental pulp cells, particularly important properties for the treatment of nerve damage, such as the expression of the GABRB1 gene, the resistance to reactive oxygen species, or both. I found it important to have them together.
That is, the present invention provides, in one embodiment, a therapeutic implant for nerve injury, comprising dental pulp cells expressing the GABRB1 gene, having reactive oxygen resistance, or having both properties. .. A preferred embodiment is a transplant material for treating nerve injury, which comprises dental pulp cells expressing the GABRB1 gene and having resistance to active oxygen. In addition, another form of the present invention includes FGF2-treated dental pulp cells in which the GABRB1 gene is expressed, has active oxygen resistance, or has both properties. , Provide therapeutic implants for nerve injury.

As used herein, the term "pulp cell" refers to a cell collected from the pulp tissue of a deciduous tooth or a permanent tooth and includes a pulp stem cell. The dental pulp cells used in the present invention are not limited as long as they have an effect of improving the function of nerve injury when transplanted to a nerve injury site, and even cells directly collected from a living body are cultured cells. It may be a cell that has been thawed after cryopreservation. As a preferred embodiment, all or most of the dental pulp cells used in the transplant material for treating nerve injury of the present invention may be dental pulp stem cells.
Therefore, the present invention provides, as an embodiment, a transplant material for treating nerve injury, which is a dental pulp stem cell treated with FGF2 and contains a dental pulp stem cell expressing the GABRB1 gene. In addition, the invention disclosed in the present specification can be read as "pulp stem cell" as described above in a preferred embodiment.

As used herein, the term "pulp stem cell" is a type of tissue stem cell that can be isolated from dental pulp tissue. Tissue stem cells are also called somatic stem cells, and while embryonic stem cells can differentiate into any cell, tissue stem cells are limited in the types of cells that can differentiate. Dental pulp stem cells are characterized, for example, by the surface antigen STRO-1. In addition, it is also possible to identify dental pulp stem cells using neural crest cell markers such as Nestin, SOX10, and SOX11 as indicators.

Here, the "pulp tissue" containing dental pulp cells can be collected from both deciduous teeth and permanent teeth, and can be obtained from the pulp of deciduous teeth and wisdom teeth that have been conventionally treated as medical waste. be. The pulp tissue can be extracted from a dentally extracted tooth in a dental facility and may be extracted from a spontaneously extracted tooth. A method for removing pulp tissue from a tooth is known, and a person skilled in the art can appropriately carry out the method. If the tooth cannot be frozen immediately on the spot, such as a tooth extracted by dental treatment, the tooth is immersed in a medium such as α-MEM for transportation at a low temperature (for example, 4). Can be stored at ° C). Preparation and preservation of dental pulp cells are described in Takeda, T. et al .: J. Dent. Res., 87: 676-681, 2008; Tamaki et al., J Dent Res. 2010 89: 773-778 and the like. It can be done according to the method.

Specifically, the method for isolating dental pulp cells from dental pulp tissue can be carried out, for example, as follows. After shredding the pulp tissue using a scalpel or the like, the shredded pulp tissue is enzymatically treated with dispase, collagenase, or a mixed enzyme solution thereof. After the enzyme treatment, the mixture is sufficiently mixed with a culture solution containing serum, and then impurities are removed using a cell strainer or the like. After that, the pulp cells can be collected from the pulp tissue by centrifuging (for example, 2,000 rpm, 4 ° C), discarding the supernatant, and adding the culture solution. As described above, a method for collecting dental pulp cells from dental pulp tissue is known, and those skilled in the art can appropriately set reagents and test conditions to be used.
In addition, dental pulp cells isolated from dental pulp tissue contain dental pulp stem cells in the cell population. In the present invention, when treating with FGF2, the dental pulp cell group containing dental pulp stem cells can be used as it is and cultured in a medium containing FGF2. Alternatively, FGF2 treatment may be performed after separating dental pulp stem cells from a cell population derived from dental pulp tissue by a known method using the above-mentioned cell surface marker or the like.
The origin of the dental pulp tissue is not limited to humans, and may be other mammals (for example, mice, rats, rabbits, dogs, cats, monkeys, sheep, cows, horses).

The dental pulp stem cells used in the transplant material for treating nerve injury of the present invention are isolated from dental pulp tissue and then treated with FGF2 by culturing in a medium containing FGF2-. By treating the dental pulp cells with FGF2, it is possible to obtain dental pulp cells in which the expression of the GABRB1 gene is enhanced and suitable for a transplant material for treating nerve injury.
Here, the "GABRB1 gene" is a gene encoding gamma-aminobutyric acid type A receptor beta1 subunit.
In the present specification, when the dental pulp cell is "expressed with the GABRB1 gene", it has a significantly increased expression level of the GABRB1 gene as compared with the expression level of the GABRB1 gene in the dental pulp cell not treated with FGF2. It means that you are. The dental pulp cells isolated from the dental pulp tissue are in a state where the expression of the GABRB1 gene is not enhanced unless specific conditions such as culturing in the presence of FGF2 are passed. In addition, the expression level of the GABRB1 gene is enhanced by culturing dental pulp cells in the presence of FGF2 for a certain period of time. In particular, the GABRB1 gene is the gene whose expression is most upregulated in the presence of FGF2. When the expression of the GABRB1 gene is enhanced by a method other than FGF2 treatment, the expression level of the GABRB1 gene is significantly increased as compared with the expression level of the GABRB1 gene in the cells before the application of the method. Means that.

Further, in the present specification, when "the GABRB1 gene is expressed in the dental pulp cell", the expression of the GABRB1 gene in the dental pulp cell is not limited to the following, but for example, as a preferred embodiment in the human dental pulp cell, the expression of the GABRB1 gene in the dental pulp cell is performed. The amount has an increased expression level of about 10 times or more, about 20 times or more, about 100 times or more, more preferably about 200 times or more, as compared with the expression level of the GABRB1 gene in FGF2-treated dental pulp cells. It is a thing.
Depending on the donor from which it was derived, transplantation of a therapeutic material for transplantation containing dental pulp cells may not be effective in improving nerve damage. When transplanted to a site, it has an excellent therapeutic effect on nerve damage.

Further, in one embodiment of the dental pulp cells used for the transplant material for treating nerve damage of the present invention, in addition to the GABRB1 gene, "MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, SCG2 gene, And at least one gene selected from the group consisting of NTSR1 genes "is expressed. The "MMP1 gene" is a gene encoding Matrix metallopeptidase 1 (interstitial collagenase). The "DRD2 gene" is a gene encoding Dopamine receptor D2. The "ABCA6 gene" is a gene encoding ATP-binding cassette, sub-family A (ABC1), member6. The "TMEM100 gene" is a gene encoding Transmembrane protein 100. The "THBD gene" is a gene encoding thrombomodulin. The "NTSR1 gene" is neurotensin receptor 1. The "SCG gene" is a gene encoding Secretogranin II.
In the present specification, when "at least one gene selected from the group consisting of MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, SCG2 gene, and NTSR1 gene is further expressed", GABRB1 In addition to the expression of a gene, it means that one, two, three, four, five, six, or seven genes in the above-listed gene group are expressed.

Specific examples of the dental pulp cells expressing one of the genes listed above in addition to the GABRB1 gene include dental pulp cells expressing the GABRB1 gene and the MMP1 gene, the GABRB1 gene and the DRD2. Dental cells expressing the gene, dentin cells expressing the GABRB1 gene and the ABCA6 gene, dentin cells expressing the GABRB1 gene and the TMEM100 gene, and the GABRB1 gene and the THBD gene are expressed. These are the dental pulp cells that are present, the dental pulp cells that express the GABRB1 gene and the SCG2 gene, and the dental pulp cells that express the GABRB1 gene and the NTSR1 gene. Also, when two, three, four, five, six, or seven genes in the above-listed gene group are expressed, the MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, All combinations of two genes selected from the group consisting of THBD gene, SCG2 gene, and NTSR1 gene, all combinations of three genes, all combinations of four genes, all combinations of five genes, Includes all combinations of six genes or all combinations of seven genes.

In the present specification, when the genes listed above are expressed, it means that the gene expression is enhanced as compared with the expression level of the corresponding gene in the dental pulp cells not treated with FGF2. .. Preferably, it means that the gene expression level is significantly increased as compared with the expression level of the corresponding gene in the dental pulp cells not treated with FGF2. The genes whose gene expression is significantly increased compared to the corresponding gene expression in FGF2-treated dental pulp cells are the MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, and SCG2 gene. be. Therefore, for example, a dental pulp cell expressing the MMP1 gene is a dental pulp having a significantly increased expression level of the MMP1 gene as compared with the expression level of the MMP1 gene in the dental pulp cells not treated with FGF2 in a preferable form. Refers to cells. Similar to the GABRB1 gene, the MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene are also cultured in the presence of FGF2 in the dental pulp cells isolated from the dental pulp tissue. Unless a specific condition such as is passed, the expression is not enhanced. In addition, the expression level of these genes is enhanced by culturing dental pulp cells in the presence of FGF2 for a certain period of time.

Hereinafter, the preferable morphology of the dental pulp cells in which the expression of each gene is enhanced will be specifically described by taking human dental pulp cells as an example. However, the dental pulp cells of the present invention are not limited to the following as long as the expression of each gene is enhanced.
In the preferred embodiment, the dental pulp cells in which the expression of the MMP1 gene is enhanced by the FGF2 treatment is the MMP1 gene whose expression level is increased by about 145 times or more as compared with the expression level of the MMP1 gene in the dental pulp cells not treated with the FGF2. Has an expression level.
Further, in the dental pulp cells in which the expression of the DRD2 gene is enhanced by the FGF2 treatment, in the preferred embodiment, the expression level of the DRD2 gene in the dental pulp cells not treated with the FGF2 is increased by about 104 times or more. It has the expression level of the gene.
Further, in the dental pulp cells in which the expression of the ABCA6 gene was enhanced by the FGF2 treatment, in the preferred embodiment, the expression level of the ABCA6 gene in the dental pulp cells not treated with the FGF2 was increased by about 17 times or more. It has the expression level of the gene.
Further, in the dental pulp cells in which the expression of the TMEM100 gene is enhanced by the FGF2 treatment, in the preferred embodiment, the expression level of the TMEM100 gene in the dental pulp cells not treated with the FGF2 is increased by about 54 times or more. It has the expression level of the gene.
Further, in the dental pulp cells in which the expression of the THBD gene is enhanced by the FGF2 treatment, in the preferred embodiment, the THBD increased by about 6 times or more as compared with the expression level of the THBD gene in the dental pulp cells not treated with the FGF2. It has the expression level of the gene.
Further, in the dental pulp cells in which the expression of the NTSR1 gene is enhanced by the FGF2 treatment, in the preferred embodiment, the SCG2 gene is increased by 8 times or more as compared with the expression level of the NTSR1 gene in the dental pulp cells not treated with the FGF2. Has an expression level of.
Further, in the dental pulp cells in which the expression of the SCG2 gene is enhanced by the FGF2 treatment, in the preferred embodiment, the expression level of the SCG2 gene in the dental pulp cells not treated with the FGF2 is increased by about 5 times or more. It has the expression level of the gene.

Expression of the GABRB1 gene, MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene can be measured using known methods, such as real time PCR, Northern blotting, In situ hybridization, RNAse protection assay, reverse transcription polymerase chain reaction (RT-PCR) and the like can be mentioned.
Regarding the expression level of each gene, when comparing the expression level of the gene in the dental pulp cells not treated with FGF2, the following Example 2-2. It is preferable to follow the method described in Real-time PCR.
Specifically, the reaction conditions for Real-time PCR are 40 cycles with 95 ° C for 30 seconds as one cycle, 95 ° C for 5 seconds, and 60 ° C for 30 seconds as one cycle, using primers corresponding to each gene. Perform one cycle of 15 seconds, 60 ° C for 30 seconds, and 95 ° C for 15 seconds.
In addition, by using the expression of these genes as an index, it is possible to screen dental pulp cells suitable for a transplant material for treating nerve injury. In particular, since the enhancement of GABRB1 gene expression by FGF2 treatment correlates with the therapeutic effect, it is preferable to use the enhancement of GABRB1 gene expression as an index.

That is, in one embodiment, the present invention is a method for screening dental pulp cells suitable for a transplant material for treating nerve damage, measuring the expression of the GABRB1 gene in the FGF2-treated dental pulp cells, and expressing the GABRB1 gene. Methods involving the selection of pulp cells that are present are included. In addition, one embodiment of the screening method is at least one selected from the group consisting of the MMP1 gene, the DRD2 gene, the ABCA6 gene, the TMEM100 gene, the THBD gene, the NTSR1 gene, and the SCG2 gene in addition to the GABRB1 gene. It includes a form in which the expression of a gene is measured and the dental pulp cells expressing the measured gene are selected. By screening FGF2-treated dental pulp cells using these genes as indicators, for example, it becomes possible to select dental pulp cells having a therapeutic effect on nerve injury from among dental pulp cells derived from a plurality of donors.

Further, in the present specification, "having resistance to active oxygen" means that dental pulp cells have resistance to toxicity of active oxygen. In particular, when dental pulp cells are used as a transplant material, it means that they have resistance to the toxicity of active oxygen generated in the tissue at the damaged site of the transplant destination. The dental pulp cells having active oxygen resistance of the present invention have a higher cell engraftment rate after transplantation than the dental pulp cells not having active oxygen resistance, and have an excellent effect on improving the function of the injured site. be.
Here, the active oxygen tolerance of dental pulp cells is not limited to the following, but for example, hydrogen peroxide (H ₂ O ₂ ) is added to the medium in which dental pulp cells are cultured and cultured, and then by MTT assay or the like. Can be evaluated.

In the present specification, when referred to as "dental pulp cells having resistance to active oxygen", in one preferred embodiment, any of the following conditions a) to f) is satisfied:
A) The living dental pulp cells at the time of culturing in 10% FCS-αMEM medium containing 0.5 mM hydrogen peroxide for 24 hours were cultured in 10% FCS-αMEM medium without adding hydrogen peroxide for 24 hours. It survives at a rate of about 50% or more, preferably about 60% or more, more preferably about 70% or more, and further preferably about 80% or more with respect to the number of viable dental pulp cells.
B) The living dental pulp cells at the time of culturing in 10% FCS-αMEM medium containing 0.6 mM hydrogen peroxide for 24 hours were cultured in 10% FCS-αMEM medium without adding hydrogen peroxide for 24 hours. It survives at a rate of about 40% or more, more preferably about 50% or more, still more preferably about 60% or more with respect to the number of viable dental pulp cells.
C) The living dental pulp cells at the time of culturing in 10% FCS-αMEM medium containing 0.7 mM hydrogen peroxide for 24 hours were cultured in 10% FCS-αMEM medium without adding hydrogen peroxide for 24 hours. It survives at a rate of about 15% or more, more preferably about 20% or more, still more preferably about 25% or more with respect to the number of viable dental pulp cells.
D) The living cells of the dental pulp cells when cultured in 10% FCS-αMEM medium containing 0.5 mM hydrogen peroxide for 24 hours are the living cells when the dental pulp cells not treated with FGF2 are cultured for 24 hours under the same conditions. Surviving at a rate of about 3 times or more, more preferably about 4 times or more, still more preferably about 5 times or more with respect to the number.
E) Live cells of dental pulp cells cultured in 10% FCS-αMEM medium containing 0.6 mM hydrogen peroxide for 24 hours, and live cells of dental pulp cells not treated with FGF2 for 24 hours under the same conditions. Surviving at a rate of about 3 times or more, more preferably about 4 times or more, still more preferably about 5 times or more with respect to the number.
F) Live cells of dental pulp cells cultured in 10% FCS-αMEM medium containing 0.7 mM hydrogen peroxide for 24 hours, and live cells of dental pulp cells not treated with FGF2 for 24 hours under the same conditions. It survives at a rate of about 3 times or more, more preferably about 4 times or more, still more preferably about 5 times or more with respect to the number.

The method of FGF2 treatment for using dental pulp cells as a therapeutic implant for nerve injury can be performed, for example, according to the method described in International Publication No. 2014/185470. Specifically, but not limited to the following embodiments, FGF2 treatment is performed by, for example, culturing dental pulp cells isolated from dental pulp tissue in a medium containing FGF2 for a certain period of time.

Here, in the present specification, the "medium containing FGF2" is, for example, a medium in which FGF2 is added as a growth factor to a basal medium containing serum; a medium in which FGF2 is added to a basal medium containing no serum. FGF2 added to the basal medium containing serum; FGF2 added as a growth factor to a medium marketed as a medium for culturing mesenchymal stem cells; commercially available as a medium for culturing mesenchymal stem cells Examples of the medium include a medium to which FGF2 has been added.

Further, in the present specification, FGF2 means a basic fibroblast growth factor (FGF) and is also referred to as bFGF or HBGF-2.
As FGF2, a commercially available product can be appropriately diluted before use. Since it is used as a transplant material, it is preferably filtered with an appropriate membrane and confirmed to be negative for bacteria, fungi, mycoplasma and the like. The concentration of FGF2 is not particularly limited as long as the obtained transplant material has a sufficient therapeutic effect on spinal cord injury, but can be, for example, 5 ng / ml or more, 7 ng / ml or more, and 10 ng / ml or more. A preferred embodiment is a method of culturing dental pulp cells using a medium supplemented with FGF2 at a concentration of 10 ng / ml. Further, the FGF2 treatment is not limited to the following as long as the expression of the GARBR1 gene is enhanced, but an embodiment in which FGF2 is added to the medium every day is preferable.

As used herein, the term "basic medium" refers to a medium containing only known components having a low molecular weight, and non-limiting examples of the basal medium include BME (Basal medium Eagle's), MEM (Minimum essential medium), and DMEM (Dulbecco's modified). Eagle's medium), Eagle's medium such as RPMI1630, RPMI1640, RPMI (Roswell Park Memorial Institue) medium, Fisher's medium, F10 medium, Ham's medium such as F12 medium, MCDB104, 107, 131, 151, MCDB media such as 153, 170, 202 and RITC80-7 medium are known and can be appropriately selected.

As used herein, the term "medium marketed as a medium for culturing mesenchymal stem cells" means a commercially available medium for culturing and proliferating mesenchymal stem cells in a state where differentiation ability is maintained without inducing differentiation. , For example, MSCGM medium (LONZA), mesenchymal stem cell growth medium (Takara Bio Co., Ltd.), mesenchymal stem cell growth medium DXF (Takara Bio Co., Ltd.), Stemline (registered trademark) mesenchymal stem cell growth medium (Sigma). -Aldrich), MF-medium ™ mesenchymal stem cell growth medium (Toyo Spinning Life Science), BD Mosaic ™ mesenchymal stem cell serum-free culture kit (BD bioscience). Not limited.

As used herein, the term "serum" is not limited as long as it is a serum used for cell culture, and examples thereof include human serum, fetal bovine serum, and horse serum. When the therapeutic transplant material according to the present invention is transplanted into a human, it is preferably human serum. The serum is preferably less than 15% by weight, less than 13% by weight, less than 10% by weight, less than 8% by weight, less than 5% by weight, etc. in the medium.

Further, as one embodiment in the step of culturing dental pulp cells using a medium containing FGF2, "a medium substantially free of growth factors other than FGF2" can be used. As used herein, the term "medium substantially free of growth factors other than FGF2" means that FGF2 is the only growth factor intentionally added.

As used herein, "growth factor" means various proteins called growth factors or growth factors, for example, epithelial growth factor (EGF), fibroblast growth factor (FGF), acidic fibroblast growth factor (aFGF or FGF1), basic fibroblast growth factor (bFGF or FGF2), platelet-derived growth factor (PDGF), nerve growth factor (NGF), insulin-like growth factor (IGF), hepatocellular growth factor (HGF), transforming growth Factors (TGF), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) interleukins and the like.

In addition, other substances useful for cell culture can be appropriately added to the medium used in the method for producing a transplant material for treating spinal cord injury according to the present invention. Such substances include, for example, buffers for stabilizing pH (HEPES, etc.), phenol red pH indicators, antibiotics (penicillin G, streptomycin, ampicillin B, gentamicin, kanamycin, ampicillin, minomycin, gentamicin, etc.), amino acids, Examples include, but are not limited to, vitamins, lipids, sugars, nucleic acids, inorganic salts, organic acid salts, minerals, and the like.

In the method for producing a transplant material for treating nerve injury according to the present invention, it is also preferable to subculture the dental pulp cells twice, three times, four times, five times, or six times or more in the above-mentioned medium. In order to impart active oxygen resistance to dental pulp cells by FGF2 treatment, it is preferable to carry out a period of at least two subcultures or a culture of at least 6 days. Even dental pulp cells treated with FGF2 lose their resistance to active oxygen by subculturing for 1 to 2 passages in a medium containing no FGF2 or culturing in a medium containing no FGF2 for about 3 days. Therefore, although it is not limited as long as the dental pulp cells exert a therapeutic effect on nerve damage, it is more preferable to culture using a medium containing FGF2 until immediately before using the dental pulp cells as a therapeutic transplant material.
The culturing method is not particularly limited except for culturing in a medium substantially free of growth factors other than FGF2, and those skilled in the art can perform various conditions (temperature, humidity, CO ₂ concentration) depending on the type of cells to be cultivated. , PH, frequency of medium replacement, etc.) can be selected.

In the method for producing a transplant material for treating nerve injury according to the present invention, in addition to the above-mentioned culture step, various steps suitable as a method for producing the transplant material can be performed. For example, the culture obtained in the culture step may be mixed with a highly viscous substance such as hyaluronic acid, collagen gel, fibrinogen, soft agar, or a synthetic polymer to adjust the fluidity. By imparting appropriate fluidity, the transplant material can be fixed to the damaged site.
After mixing with a gel such as collagen gel, soft agar, or synthetic polymer, the culture may be performed for a certain period of time to perform three-dimensional culture.

As used herein, "nerve injury" means central and peripheral nerve injury, specifically spinal cord injury, cerebral infarction, intracerebral hemorrhage, submucosal hemorrhage, spinal cord hemorrhage, nerve compression injury due to herniated disc, sciatic bone. It includes, but is not limited to, nerve pain, peripheral neuropathy due to diabetes, and the like. The transplant material of the present invention can be applied to any nerve injury as long as the therapeutic effect can be obtained by transplantation. The therapeutic effect is not limited to the effect of curing the disease, but includes the effect of improving at least one symptom associated with the disease, the effect of preventing or delaying the progression of the disease, and the like.

The effect of the therapeutic transplant material obtained by the method for producing a therapeutic transplant material for nerve injury according to the present invention can be evaluated, for example, by performing cell transplantation on a nerve injury model animal.
For example, it can be evaluated using a rat spinal cord injury model prepared by a known method. Specifically, the rat is under anesthesia such as isoflurane, and after laminectomy, the spinal cord is completely amputated using a surgical scalpel. The cutting site can be, for example, the 10th thoracic vertebra (Th10). Hemostasis is performed after cutting the spinal cord, and then a medium containing dental pulp cells is transplanted to the injured site using a syringe. For example, 10 μl of a medium containing cells at a concentration of about 1 × 10 ⁶ is transplanted to the injured site. After injecting the cells, let stand for about 10 minutes and close the wound with sutures. Rats may be placed in the warming chamber until awake after surgery. In addition, an anesthetic antagonist may be administered as needed. After surgery, antibiotics and immunosuppressants may be administered as needed.
For example, at 7 weeks after transplantation, improvement of motor function is evaluated, tissue repair by immunohistochemical staining of dental pulp cell transplantation site is evaluated, and functional recovery of damaged site is evaluated by electrophysiological method for treatment. Confirm the effect of the transplant agent.

The evaluation of motor function can be evaluated by the BBB score (Basso DM et al., J Neurotrauma. 1995 Feb; 12 (1): 1-21).
In addition, immunohistochemical staining can be performed on frozen sections of the spinal cord injury site prepared by a known method. Specifically, rats are perfused and fixed transcardially under anesthesia, and spinal cord tissue is collected. Tissues are frozen and embedded with an embedding agent and sections are prepared with a cryostat. For immunostaining, anti-growth-associated protein (GAP) 43 antibody, which is a growth cone marker, anti-glial fibrillary acidic protein (GFAP) antibody, which is an astrocyte marker, anti-GFP antibody, and anti-MBP antibody, which is an oligodendrocyte marker, were used. Can stain the tissue. When dental spinal cells are transplanted to the nerve injury site, the regenerated axons of GAP43-positive cells are elongated through the injured site, and the regenerated axons that are myelinated so that the axons are surrounded by the MBP-positive cells can be confirmed. Or, it is preferable that GFAP-positive endogenous astrocytes are preserved as a scaffold for the regenerated axon.
In addition, for example, when dental pulp cells are transplanted to a nerve injury site, in a preferable form, one week after the dental pulp cell transplantation, GAP-43-positive spinal cord regeneration fibers are three times as much as those in the control group not transplanted with dental pulp cells. As described above, preferably 5 times or more, more preferably 7 times or more are recognized.
In addition, evaluation by an electrophysiological method can be performed by measuring an electrical action potential through a spinal cord amputation. For example, for rats in which the Th10 site has been amputated, under anesthesia, electrical stimulation is applied at Th8 via Th10, and microelectrodes are inserted into the spinal cord so that action potentials can be detected at Th13. do. The electrical stimulus can be evaluated, for example, by giving a short square wave pulse (0.2 seconds) every 2 seconds, detecting the action potential at Th13, and measuring the latency.

The transplant material for treating nerve injury according to the present invention contains the dental pulp cells in which the GABRB1 gene is expressed by the above-mentioned FGF2 treatment. As one embodiment of the method for producing a transplant material for treating nerve damage, a medium containing FGF2 in which dental pulp cells have been cultured can be used as it is in a state containing dental pulp cells, or can be used as a therapeutic transplant material. The dental pulp cells may be transferred to the medium or solution of the above and then used as a therapeutic transplant material. The transplant material may contain a gel such as collagen gel, soft agar, or a synthetic polymer, and the viscosity may be adjusted by an appropriate gelling agent or thickener.

The present invention also includes a method for treating nerve injury, which comprises a step of transplanting the above-mentioned therapeutic implant for nerve injury to a site of nerve injury.
The transplant material for treating nerve injury can be injected into, for example, a nerve injury site by a syringe or the like. In addition, the damaged site may be incised and the transplant material may be placed. If the transplant material contains allogeneic cells, an immunosuppressive agent such as cyclosporine may be administered at the same time. It can be used in combination with other drugs as long as it has a therapeutic effect on nerve damage.
The dose and the number of administrations can be appropriately determined by those skilled in the art.
The target of the treatment method for nerve injury is not limited to humans, and may be other mammals (eg, mice, rats, rabbits, dogs, cats, monkeys, sheep, cows, horses).
In the present specification, examples of treatment methods for human spinal cord injury are shown below, but the treatment methods are not limited thereto.
Spinal cord injury in a human spinal cord injury patient under anesthesia using the therapeutic implant material produced by the method for producing a therapeutic implant material for nerve injury according to the present invention or the therapeutic implant material for nerve injury according to the present invention. An effective amount of dental spinal cord cells can be directly transplanted to the site using a syringe or the like.

Further, the method for treating a nerve injury of the present invention is a method including, in one embodiment, a step of transplanting the above-mentioned therapeutic implant for nerve injury in combination with an active oxygen scavenger to a site of nerve injury. ..
As used herein, the term "active oxygen scavenger" means an agent that has the effect of preventing damage to the site of nerve damage in the living body caused by active oxygen or the transplanted dental pulp cells, and at the same time as the transplantation of dental pulp cells. , Or in combination.
Such an active oxygen scavenger is not particularly limited as long as it can be administered to a living body and has an effect of protecting transplanted cells from damage to active oxygen at a damaged site. For example, edaravone and vitamin C. , Nrf2 inducer, glutathione activity inducer and other known active oxygen scavengers can be used.
The method, dose, and frequency of administration of the active oxygen scavenger can be appropriately set by those skilled in the art depending on the active oxygen scavenger to be used and the damaged site.
For example, when edaravone is administered to spinal cord injured rats in combination with dental pulp cell transplantation as an active oxygen scavenging agent, edaravone is administered twice daily for 1 week immediately after cell transplantation surgery, but is not limited to the following. It can be administered intraperitoneally at a dose of mg / kg. It has been confirmed that edaravone alone cannot be used alone in a spinal cord injury model to obtain a significant functional recovery.
In addition, for example, when edaravone is administered to humans in combination with dental pulp cell transplantation as an active oxygen scavenging agent, it is not limited to the following, but immediately after the cell transplantation surgery, it is divided into once or twice a day, 2 Edaravone can be infused intravenously at a dose of 60 mg / kg over a week.

In addition, the method for treating nerve injury of the present invention is to transplant a transplant material for treating nerve injury to a nerve injury site in combination with an active oxygen scavenging agent. As described above, the active oxygen scavenger has an effect of preventing cell damage of active oxygen occurring at a nerve injury site. Therefore, the dental pulp cells transplanted in combination with the administration of the active oxygen scavenger do not necessarily have the active oxygen resistance. That is, the dental pulp cells used for transplantation do not necessarily have to be dental pulp cells that have acquired active oxygen resistance by FGF2 treatment.

The present invention also includes a kit for manufacturing a implant for treating nerve injury. Such a kit contains all or part of a medium for culturing dental pulp cells or a part thereof, FGF2, and a reagent for measuring gene expression (for example, a primer for GABRB1 amplification). Further, an active oxygen scavenging agent used at the time of cell transplantation may be contained. Examples of the medium for culturing dental pulp cells include a basal medium and a medium for culturing mesenchymal stem cells. FGF2 may be separate from the medium or may be mixed from the beginning. In addition, the medium should be prepared by the user, such as ultrapure water, which is always available in the laboratory, and contains all or part of the necessary components so that the medium of the present invention can be prepared simply by mixing it. May be.
The kit of the present invention may be used for laboratory experiments or may be used for mass culture. In addition to the culture solution, a culture vessel, a virus filter, a coating material for the culture vessel, various reagents, a buffer solution, an instruction manual, and the like may be provided.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. In addition, the contents of the literature cited herein are incorporated herein by reference.

The recovery and genomic / gene analysis of human dental pulp tissue described in Examples 1 and 2 below have been approved by the Gifu University Ethics Committee (approval numbers 26-116). All animal experiments strictly followed the protocol approved by the Animal Care and Use Committee of Gifu University (approval numbers 25-20, 25-68, 27-83).

<Example 1. Treatment of spinal cord injury with FGF2-treated human dental pulp cells>
So far, it has been reported that either pulp cell transplantation or FGF2 administration promoted functional recovery from a completely amputated spinal cord. To confirm these effects, human dental pulp cells were transplanted into the injured part of the spinal cord of rats, and a test was conducted to evaluate the motor function of the hind limbs (BBB score) in combination with an electrophysiological test.
It has also been confirmed that the efficiency of iPS induction differs depending on the donor from which the developing tooth used to prepare human dental pulp cells is derived. In this example, the improvement effects of spinal cord injury after transplantation were compared using each human dental pulp cell lineage obtained from four donors of different ages and genders shown in the table below.

なお、具体的には、下記方法により行った。

Specifically, it was carried out by the following method.

<1-1. Isolation and culture of human dental pulp cells and viral infection>
The present inventors collected approximately 300 DPC strains at the University Hospital attached to Gifu University School of Medicine. Patient-derived human dental pulp cells provided based on informed consent were isolated and cultured by a known method (Gronthos et al., 2000, Takeda et al., 2008).
Lentiviruses for gene transfer are available as lentiviral vectors (EF-1α promoter, Venus) (Supplementary Table S.) and plasmids (pLP / VSV-G, pLP1, pLP2) (Miyoshi, H. et al., Development of a). It was produced in a HEK293FT cell line using a self-inactivating lentivirus vector. J. Virol. 1998). The lentiviral vector contains a sequence in which the Venus gene is bound downstream of the EF1α promoter.
Human dental pulp cells are cultured at 37 ° C. in MSCGM medium (Lonza) in 21% O ₂ , 5CO ₂ humidified air and infected with lentivirus to trace the cells in transplantation at 5-6 passages. rice field. After lentivirus infection, human dental pulp cells were cultured in α-MEM medium (Sigma). The efficiency of virus infection was confirmed using FACS.
In addition, FGF2 (R & D SYSTEMS) was added to α-MEM medium at a concentration of 10 ng / μl daily, and human dental pulp cells treated with FGF2 were used for transplantation in the 10th to 13th generations of passage. The dental pulp cell transplantation groups derived from each donor treated with FGF2 are referred to as DP1-FGF (+) group, DP31-FGF (+), DP165-FGF (+), and DP296-FGF (+), respectively. In addition, as a control group for the addition of FGF2, a subculture of DP1 or DP31 was prepared in α-MEM medium containing no FGF2, and used for transplantation in the 10th to 13th generations of the passage (DP1 respectively). -FGF (-) group or DP31-FGF (-) group).

<1-2. Animal and transplant surgery>
Female Wistar adult rats (148g-168g) were purchased from Nippon SLC Co., Ltd. Rats are placed under appropriate anesthesia by inhalation of 1.5-2% isoflurene (TK-7, Biomachinery Co., Ltd., Japan), and after laminectomy, the 10th thoracic spinal cord (Th10) is surgically scalpel (Surgical). The spinal cord was completely cut by cutting exactly twice slowly using blade stainless steel no.11 mini, kai medical, Japan). The cutting was visually confirmed. After hemostasis, a medium containing cells at a concentration of about 1 × 10 ⁶ in 10 μl medium was carefully and slowly injected into the injured site using a Hamilton syringe (Sigma-Aldrich, Missouri, USA). After cell injection, the cells were left untouched for 10 minutes. After leaving for 10 minutes, the wound was closed with sutures. In addition, instead of injecting cells, the same amount of PBS was added to the spinal cord amputation site in the rats in the control group, and after standing for 10 minutes in the same manner, the wound was closed by suturing (PBS administration group). Rats were placed in a rewarming chamber until waking up after surgery.

Then, antibiotics (sulbactam / ampicillin, SANDOZ, 10 mg / kg body weight) were administered daily for 1 week. In addition, immunosuppressive agents ((cyclosporine, Novartis, 10 mg / kg body weight) were administered daily from the day before surgery to the rats in the control group (PBS administration group) to which human dental pulp cells were transplanted. The experimental animals are bred in an environment where water can be easily supplied by a custom-made water supply facility equipped with a long nozzle, can be fed at all times, and are illuminated at 26 ° C, 65% humidity, and 12 hours a day. It was a condition.

<1-3. Transplantation of human dental pulp cells into the rat vitreous cavity>
In order to investigate the carcinogenicity of human dental pulp cells treated with FGF2, the above 1-1. Human dental pulp cells (DP31) cultured in an FGF2-containing medium were transplanted into the rat vitreous cavity by the method described in 1.
Specifically, rats were anesthetized with 1.5-2% isoflurene and the rat cornea was further anesthetized with 4% oxybuprocaine (Benoxil, Santen). A hole was made in the cornea using a 27-gauge hypodermic needle (terumo, USA). The hypodermic needle was then removed and a 30 gauge blunt needle (Hamilton Company, Reno, NV, USA) was inserted into the anterior chamber through the hole created and avoiding the glow and lens. The needle was inserted toward the retinal surface through the vitreous cavity, and after the needle reached the retinal surface, 10 μl of DMEM medium containing DP31 treated with FGF2 was injected into the vitreous cavity. After injecting the cells, one drop of ofloxacin ophthalmic solution (Tarivid topical solution, Santen) was instilled into the eye. Six rats were used in this study, two of which were transplanted with 0.5 x ¹⁰⁶ cells and the remaining four were transplanted with 4 x ¹⁰⁶ cells. All rats received immunosuppressive drugs ((cyclosporine, Novartis, 10 mg / kg body weight) daily from the day before surgery to daily after surgery.

<1-4. BBB score>
Rats were videotaped for 30 seconds to 2 minutes each week after surgery to objectively analyze the motor function of the rats after surgery. Population identity was ruled out by independent observers who had previously performed BBB score analysis, and the BBB locomotion rating scale was assessed by watching the video (Basso et al., 1996). Detailed evaluation criteria are shown in Fig. 1 below.

<1-5. Electrophysiological test>
In rats after human dental pulp cell transplantation, an electrophysiological test was performed by the following method in order to confirm whether it is possible to transmit an electrical action potential through a spinal cord cut.
The rat was anesthetized with urethane and the rat organ was incised to open the airway. The transmission of electrical activity through the graft was evaluated using the following method.
In this test, a bespoke bipolar electrode equipped with tungsten microelectrodes (φ0.2 mm, Unique Medical, Japan.) At 1 mm intervals was used. The electrodes were inserted 1.0 m below the center of the spinal cord and 0.5-0.75 mm laterally to stimulate the Th8 spinal cord segment and record electrical activity in the Th13 spinal cord segment. To insert microelectrodes, fix the rat using a spinal fixation device (ST-7R-HT, NARISHIGE, Tokyo, JAPAN), control the microelectrodes with a manipulator, and confirm the position of the microelectrodes under a surgical microscope. I went by doing. As an electrical stimulus, a short square wave pulse (0.2 seconds) was given every 2 seconds.

<1-6. Immunohistochemistry>
Eight weeks after surgery for immunohistochemical staining, rats were anesthetized and fixed by transcardiac perfusion with 1% PBS and 4% paraformaldehyde. Spinal cord tissue was removed, further post-fixed with 4% paraformaldehyde for 24 hours, and then placed overnight in 30% sucrose solution. Then, the spinal cord was used as a frozen section in an OCT compound (Sakura Finetek), and a 25 mm sagittal section frozen section was prepared using a cryostat (CM3050S, Leica). Frozen sections were prepared as primary antibodies such as anti-GFAP antibody (rabbit IgG, 1: 500, abcam), anti-GAP-43 antibody (mouse IgG, 1: 200, Millipore), and anti-GFP antibody (rabbit IgG, 1: 200, Millipore). ), Anti-MBP antibody (rabbit IgG, 1: 200, Millipore) was reacted. It is then stained with anti-rabbit IgG-Alexa 546, anti-mouse IgG-Alexa Fluor 488, anti-mouse IgG-Alexa 546, and anti-rabbit IgG-Alexa Fluor 488 as secondary antibodies. Stained with DAPI (Sigma-Aldrich). Images of stained sections were obtained using a fluorescence microscope (BZ-9000, Keyence).

<1-7. Result>
As a result, the group transplanted with DP1 (DP1-FGF2 (-) group) and DP31 (DP31-FGF2 (-) group) cultured without adding FGF2 had significant motor function as compared with the PBS-administered group. No recovery could be observed (Fig. 2a). Similarly, of the 6 animals transplanted with DP1-FGF2 (-) and DP31-FGF2 (-), electrical action potentials could be detected through the completely cut site 8 weeks after spinal cord injury. There was only one (Fig. 2b). Next, when a similar test using α-MEM containing 10 μg / ml FGF2 (FGF2 single administration group) was attempted, the function was slightly improved compared to the PBS administration group without the immunosuppressant. Only shown (Fig. 3a). In the FGF2 single administration group, action potentials could be detected from only one of the four animals (Fig. 3b).
On the other hand, the group to which DP1 or DP31 cultured in the FGF2-containing medium (DP1-FGF2 (+) group or DP31-FGF2 (+) group) showed significant recovery after spinal cord injury in both groups. An improvement in action potential was seen (Fig. 4).

Human dental pulp cells expressing Venus protein containing a nuclear translocation signal were transplanted to confirm that human dental pulp cells treated with FGF2 were not maintained for a long time at the site after transplantation and disappeared immediately from the site of spinal cord injury. .. As a result, almost no fluorescence was detected at the injured site at the time point 8 weeks after the spinal cord injury. To support this result, when the same human dental pulp cells were transplanted into the vitreous cavity, the split surface of the vitreous was observed with a fluorescence microscope (SZX-RFL2, OLYMPUS) 3 weeks after the transplantation. , Fluorescence could not be detected (Fig. 5). This indicates that the transplanted cells were not maintained long enough to be detected in the spinal cord injury model system of this study, and no tumorigenesis of the transplanted cells was observed (Note that Examples). In the result of 4, the FGF2-treated dental pulp cells remained slightly after 7 weeks of transplantation and were not detected. Although the detection power was different due to the difference in the structure of the lentivirus vector, the two results were obtained. All indicate that most of the transplanted cells are not maintained in the transplanted spinal cord tissue.)

Moreover, when the therapeutic effect was compared for each donor from which it was derived, the group transplanted with DP1, DP31 or DP165 cultured in the FGF2-containing medium (DP1-FGF (+) group, DP31-FGF2 (+) group, or In the DP165-FGF2 (+) group), a significant improvement in motor function was observed in the BBB score in all the groups as compared with the control group (PBS-administered group) (Fig. 4a). Interestingly, however, the same effect could not be confirmed in the group transplanted with DP296 cultured in the FGF2-containing medium (DP296-FGF2 (+) group) (FIG. 4a).

To support this, in the DP296-FGF2 (+) group, when an electrical stimulus of 0.6 mA was applied, the DP1-FGF (+) group, DP31-FGF2 (+) group, or DP165-FGF2 The latency was significantly prolonged (6.14 ± 1.4ms) compared to the latency (4.24 ± 0.38ms, 3.67 ± 0.34ms, 3.72 ± 0.32ms) of the three sections of the (+) section (Fig. 4b). And c).
If the spinal cord is not injured, stimulation at Th8 causes a short latency evoked potential at Th13. No action potential occurred in the PBS-administered plot. On the other hand, in the groups transplanted with DP1, DP31, and DP165, recovery of the evoked response with prolonged latency was observed. In addition, in DP296, the action potential could be observed as described above, but the latent response was significantly prolonged when compared with the group without spinal cord injury.

Figure 6 shows the results of spinal cord immunohistochemistry at a time point 8 weeks after complete dissection of the spinal cord and transplantation of human dental pulp cells. In the DP165-FGF (+) group, GAP43-positive axons were significantly increased along the endogenous GFAP-positive astrocytes (FIGS. 6a and 6b).
In addition, the regenerated axons were also involved in myelin sheath formation, and MBP-positive myelin sheaths could be confirmed (Fig. 6c). On the other hand, in the DP296-FGF (+) group, only a very small amount of regeneration was observed (Fig. 6d, e, and f). In the PBS-administered group and the DP296-FGF (+) group, most of the GFAP-positive cells disappeared in the caudal spinal cord. On the other hand, in the DP165-FGF (+) group, it was maintained even 8 weeks after the spinal cord was completely cut (Fig. 7).

<Example 2. Regulation of GABRB1 expression in human dental pulp cells by FGF2>
Comprehensive gene expression analysis was performed using DP1, DP31, DP165, and DP296 to confirm that FGF2 treatment regulates gene expression in dental pulp cells. As shown in Example 1, DP296 is a dental pulp cell that does not exert a therapeutic effect on nerve damage even by FGF2 treatment. Each human dental pulp cell used for gene expression analysis is described in 1-1. FGF2 (R & D SYSTEMS) was added to α-MEM medium at a final concentration of 10 ng / μl daily to prepare human dental pulp cells treated with FGF2 (FGF2 treated group). In addition, as the FGF2 non-added group, human dental pulp cells isolated in the same manner were cultured in an α-MEM medium containing no FGF (FGF2 untreated group). For each cell, cells of the 10th to 13th passages were used. In addition, comprehensive gene analysis was performed using a cDNA microarray, and then specific gene expression was analyzed using the Real-time PCR method. The dental pulp cells used in this example were those that had not been gene-introduced with a lentivirus.
<2-1. cDNA microarray ＞
Total RNA from cultured human dental pulp cells was isolated using the Rneasy® Plus Mini Kit (Qiagen, Valencia, CA, USA). After quantifying RNA using the Agilent 2100 Bioanalyzer, 100 ng of total RNA is reverse transcribed and amplified to cDNA using the Low Input Quick Amp Labeling kit (Agilent Technologies, Santa Clara, CA) according to the protocol. And labeled with Cy3 labeled CTP. After the labeling and purification steps, the cDNA was quantified using an ND-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE) and hybridized to a SurePrint G3 Human 8x60K v2 oligo-DNA microarray (Agilent Technologies). After hybridization, the array was washed with the Gene Expression Wash Pack (Agilent Technologies). Fluorescence images of the hybridized array were obtained with an Agilent DNA microarray scanner, and the fluorescence intensity was determined using Agilent Feature Extraction software ver.10.7.3.1. Each sample was analyzed once. The level of gene expression was determined using Gene Spring GX12.6 (Agilent Technologies).

<2-2. Real-time PCR ＞
Total RNA from cultured human dental pulp cells was isolated using the Rneasy® Plus Mini Kit (Qiagen, Valencia, CA, USA). For real-time PCR, PCR products consisting of cDNA were prepared using SYBR Premix Ex Taq (Takara, Shiga, Japan) and Thermal Cycler Dice Real-Time System (Takara). The primers used are shown in Table 2.

<2-3. Result>
As a result of cDNA microarray analysis, there were 131 genes whose expression was significantly increased by 5 times or more by FGF2 treatment in the FGF-treated group and the FGF-untreated group. In particular, GABRB1 showed a 357-fold increase in expression by treatment with FGF2. The table below is a list of genes whose expression was significantly increased 5 times or more by FGF2 treatment in comparison with the FGF-treated group and the FGF-untreated group.

Here, a real-time PCR assay was performed to assess the expression levels of GABRB1 in DP1, DP31, DP165, and DP296. As a result, as expected, in DP1, DP31, and DP165, the expression of GABRB1 was significantly increased by the treatment with FGF2, and the expression level was the highest among the genes whose expression was upregulated. However, the expression of GABRB1 in DP296 showed no significant change (Fig. 8). This result indicates that GABRB1 gene expression by FGF2 treatment can be used as an index of the therapeutic effect of nerve injury. In addition, the other seven genes upregulated by FGF2 (MMP1 gene, DRD2 gene, ABCA6 gene, TMEM100 gene, THBD gene, NTSR1 gene, and SCG2 gene) have similar expression profiles in four different cell lines. Shown (Fig. 9). Therefore, for each of these seven genes, enhanced expression is important for dental pulp cells used as therapeutic implants for nerve injury.

<Example 3. Effect of FGF2 treatment on resistance of human dental pulp cells to reactive oxygen species>
Transplantation of dental pulp cells untreated with FGF2 did not show any effective effect in improving nerve damage. Here, it is known that the concentration of active oxygen increases in the acute phase in the spinal cord injury portion. Reactive oxygen species are thought to damage not only the nervous system cells in the spinal cord but also the transplanted cells. Therefore, in order to confirm whether or not FGF2 treatment has resistance to cytotoxicity of dental pulp cells against active oxygen, the following test was conducted.

<3-1. Experimental material>
Human dental pulp tissue was collected at the Department of Oral Pathology, Gifu University School of Medicine, with the written consent of the patient to use it for research purposes. In addition, the induced and cultured dental pulp cells are cryopreserved and managed in an anonymized form that cannot be connected, and personal information is strictly protected. A research plan using the cells was submitted to the Bioethics Committees of Gifu University and Gifu Pharmaceutical University, and after receiving approval, some cells were donated from the stock and used in the experiment. The mesenchymal stem cell basal medium (MSCBM) was purchased from Lonza Japan Co., Ltd., and the α-MEM medium was purchased from Sigma Aldrich Japan Co., Ltd. Fetal calf serum (FCS) was purchased from Hyclone, and FGF-2 was purchased from R & D System.

<3-2. Culture of dental pulp cells ＞
Human dental pulp cells DP31 isolated from dental pulp tissue (that is, dental pulp cells derived from the same donor as DP31 used in Examples 1 and 2) were semiconducted into one 10 cm petri dish for 2-3 days. The cells were subcultured once (1 sheet was subcultured to 3 sheets), and up to 12 subcultures were cultured in MSCBM. Then, in order to evaluate the intratissue dynamics, the EGFP gene and puromycin resistance gene linked to the CAG promoter were introduced into dental pulp cells using a lentiviral vector, and puromycin was added and cultured to select only the gene-introduced cells. .. At this time, almost no cells that caused cell death were observed. After the gene-introduced dental pulp cells were subcultured, the dental pulp cells were cultured according to the following culture schedule.
First, in order to investigate the relationship between the timing of addition of FGF2 and the effect on resistance to active oxygen, dental pulp cells were cultured according to the culture schedules i) to iv) below. Further, the outline of the culture schedule of i) to iv) is shown in FIG.
i) Dental pulp cells subcultured in α-MEM medium containing 10% FCS (10% FCS-αMEM) for another 49 hours in 10% FCS-αMEM medium (S / S: DPC-S). Ward).
ii) 6 subcultured dental pulp cells in αMEM medium containing 10% FCS (10% FCS-αMEM) were further cultured in 10% FCS-αMEM medium for another 48 hours, and then FGF-2 (final concentration: 10). Incubate in 10% FCS-αMEM containing ng / ml) for 1 hour (S / F: DPC-S group).
iii) Dental pulp cells subcultured in 6 subcultures in 10% FCS-αMEM medium containing FGF-2 (final concentration: 10 ng / ml) and 10% FCS containing FGF-2 (final concentration: 10 ng / ml). -Culture in αMEM medium for another 49 hours (F / F: DPC-FS section).
iv) 10% FCS containing FGF-2 (final concentration: 10 ng / ml) 10% FCS containing FGF-2 (final concentration: 10 ng / ml) of dental pulp cells subcultured in 6 subcultures in MEM medium. -Incubate in αMEM medium for another 24 hours, and then incubate in 10% FCS-αMEM medium for 25 hours (F / S: DPC-FS section).
In addition, the relationship between the culture period and the effect on active oxygen resistance when the dental pulp cells cultured as the S / F: DPC-S group were cultured in the FGF2-containing medium, or the cells were cultured as the F / S: DPC-FS group. In order to investigate the relationship between the culture period and the effect on active oxygen resistance when the dental pulp cells were cultured in the FGF2-free medium, the dental pulp cells were cultured under the conditions of v) and vi) below in which the culture period was changed.
v) S / F: Dental pulp cells obtained by culturing the DPC-S group in 10% FCS-αMEM medium containing FGF-2 (final concentration: 10 ng / ml) for another 1 or 2 days. Incubate for 5 or 11 days (S / F1, S / F2, S / F5, or S / F11).
vi) F / S: The dental pulp cells obtained by culturing the DPC-FS group are further cultured in 10% FCS-αMEM medium for 1 day, 2 days, 5 days, or 11 days (F / S 1 group). , F / S 2nd ward, F / S 5th ward, F / S 11th ward).
In each of the culture schedules i) to vi), the medium was exchanged every 24 hours for the culture after the subculture.

<3-3. Resistance test to active oxygen ＞
The resistance test to active oxygen is described in 3-1. The dental pulp cells obtained by each culture schedule described in 1 above were cultured for 24 hours in a 10% FCS-αMEM medium supplemented with hydrogen peroxide (H ₂ O ₂ ) (manufactured by Wako Pure Chemical Industries, Ltd.), and then cultured. Resistance to reactive oxygen species was evaluated by performing MTT assay. In the MTT assay, MTT (manufactured by Sigma) was added to 0.5 mg / mL, reacted in a CO2 incubator for 4 hours, and then the formed formazan was dissolved in isopropanol containing 0.04M HCl to 570 nM. The absorption wavelength was measured.
Hydrogen peroxide was added to the medium in the range of 0 mM (without addition) to 0.7 mM. In addition, the condition using a medium to which hydrogen peroxide was not added was used as a control group.

<3-4. Result>
FIG. 11 shows the results of the resistance test of dental pulp cells cultured in the above i) to iv) culture schedule to active oxygen. As shown in FIG. 11, more in F / F: DPC-FS and F / S: DPC-FS than in S / S: DPC-S and S / F: DPC-S. We were able to confirm the living cells. In particular, when dental pulp cells were cultured for 24 hours in a culture medium containing hydrogen peroxide at a concentration of 0.5 mM or more, most of the cells survived in the S / S: DPC-S group and the S / F: DPC-S group. In contrast to the F / F: DPC-FS group and F / S: DPC-FS group, more viable cells could be confirmed.
In addition, the results of the resistance test of dental pulp cells cultured in the above v) and vi) culture schedules to active oxygen are shown in FIG. As shown in FIG. 12, even in the dental pulp cells of the S / F: DPC-S group, the resistance to active oxygen increased in the group cultured for 11 days in the medium containing FGF2. Although not shown, the pulp is cultured 2 to 4 times in a medium containing FGF2 (about 6 to 12 days) regardless of the culture period (1 to 7 passages) in a medium containing no FGF2. It was confirmed that the resistance of cells to active oxygen increased. On the other hand, even dental pulp cells in the F / S: DPC-FS group that acquired resistance to active oxygen lost their resistance to active oxygen by culturing in a medium containing no FGF2 for 11 days (Fig.). 12). Although not shown, resistance to active oxygen disappears by culturing dental pulp cells that have acquired resistance to active oxygen in a medium containing no FGF2 for 1 to 2 passages (about 3 to 6 days). It was confirmed.

<Example 4. Transplantation of dental pulp cells with active oxygen scavenger>
<4-1. Spinal cord injury model rat>
Wistar rats (7 weeks old, female) were purchased from Japan SLC Co., Ltd. after approval by the Animal Care and Animal Experiment Committee of Gifu Pharmaceutical University. In addition, medetomidine hydrochloride, atipamezole hydrochloride are from Nippon Zenyaku Kogyo Co., Ltd., midazolam is from Sand Co., Ltd., butorphanol tartrate is from Meiji seika Pharma Co., Ltd., water for injection is from Otsuka Pharmaceutical Co., Ltd. I bought it.

<4-2. Preparation of spinal cord injury model rat and combined use with edaravone in transplantation of dental pulp cells into spinal cord injury model rat>
Medetomidine hydrochloride, midazolam, and butorphanol tartrate were mixed to prepare a three-kind mixed anesthetic, which was intraperitoneally administered to 7-week-old female Wistar rats at a dose of 5 ml / kg. After confirming deep anesthesia by the presence or absence of a direct reflex and stimulation of the paws, a 2 cm incision was made in the back along the midline around the 10th thoracic vertebra, and fat and muscle tissue were peeled off to expose the spinal column. After that, the vertebral arch of the 10th thoracic vertebra was dissected, and the 10th thoracic spine (T10) was completely amputated with a surgical scalpel (Feather disposable scalpel mini no.14). After confirming hemostasis, 1.0 × 10 ⁶ pulp cells suspended in 10 μl phosphate-buffered saline (PBS) using a micropipette were cut into the rostral stump and tail of the cut. It was injected into the gap between the lateral stumps and the spine and skin were sutured. The dental pulp cells are described in 3-2. The cells cultured according to the culture schedule of the S / S: DPC-S group described in 1 above were used. After the operation, atipamezole hydrochloride, which is a medetomidine hydrochloride antagonist, was intraperitoneally administered at a dose of 0.75 mg / kg. Rats with a total spinal cord amputation completely lost their ability to urinate, so the bladder was stimulated to urinate twice daily during the breeding period. In addition, because it was a heterologous transplant, the immunosuppressant cyclosporine A was intraperitoneally administered once daily at a dose of 10 mg / kg body weight.
In the edaravone-administered group and the edaravone-administered cell transplantation group, edaravone (Wako Pure Chemical Industries, Ltd.) was intraperitoneally administered at a dose of 3 mg / kg twice daily for 1 week immediately after the operation.

<4-3. Frozen section preparation>
Seven weeks after cell transplantation, spinal cord injury model rats were perfused and fixed transcardially with a 4% (w / v) paraformaldehyde (PFA) solution under ether hyperanesthesia. Subsequently, the tissue was excised and fixed in the same solution at 4 ° C. overnight, and then immersed in PBS containing 20% (w / v) sucrose to replace the fixation solution (overnight). Then, it was frozen and embedded with the OCT compound of the embedding agent to form a frozen block, and stored at -80 ° C. A 25 μm-thick sagittal continuous section was prepared from the prepared frozen block using a cryostat (M1800, manufactured by Leika) and attached to a shredded MAS-coated fluorescent slide glass for the following histochemical analysis. Using.

<4-4. Immunohistochemical staining>
The reagents used for immunohistochemical staining were prepared as follows. Block Ace is Sumitomo Dainippon Pharma Co., Ltd., Vectastain ABC elite kit is Vector, MAS (Matsunami adhesive silane) coated slide glass is Matsunami Glass Co., Ltd. Mounting Medium was purchased from Thermo Fisher Scientific. Anti-growth-associated protein (GAP) 43 mouse antibody, anti-glial fibrillary acidic protein (GFAP) Rabbit antibody purchased from Chemicon, Alexa Fluor 488-labeled anti-mouse IgG antibody, Alexa Fluor 546-labeled anti-rabbit IgG antibody purchased from Molecular Probes, respectively. did.

Hereinafter, a specific method will be described. 4-3. After washing the thin sections prepared in 1 with PBS, in 0.1 M Tris-HCl buffer (pH.7.4) containing 0.3% (v / v) Triton X-100 at 37 ° C for 30 minutes, in order to increase the permeability of the antibody. Immersion treatment was performed. Tissue sections were washed with PBS and then blocked with 2% Block Ace. Then, a primary antibody that specifically recognizes GFAP, GAP-43 or GFP was added, and the mixture was reacted overnight at 4 ° C. After washing 3 times with PBS, a secondary antibody (Alexa Fluor 488-labeled anti-mouse IgG antibody or Alexa Fluor 546-labeled anti-rabbit IgG antibody) diluted 500-fold with PBS containing 2% block ace was added, and 3 at room temperature. Reacted for time. After washing with PBS three times, it was encapsulated using Perma Fluor Aqueous Mounting Medium. After encapsulation, it was sufficiently dried, and the stained image was observed and photographed using an all-in-one fluorescence microscope (KEYENCE, BZ-9000).

<4-5. Quantitative analysis of engrafted dental pulp cells in spinal cord tissue after dental pulp cell transplantation into spinal cord injury model rats>
4-3. Of the continuous sagittal sections prepared in 1 above, a total of 5 sections were prepared, one each at 0.2 and 0.4 mm on each side of the spinal cord median and on the left and right sides of the median. Then, 4-4. Immunostaining using an anti-GFP antibody was performed according to the method described in 1. The number of GFP-positive cells within a range of 2 mm from the center of injury to the rostral and caudal sides was measured, and the number of GFP-positive cells for each section was quantified.

<4-6. Evaluation of motor function of hind limbs ＞
The BBB score was used to evaluate the motor function of the hind limbs of the spinal cord injury model rat every week for 7 weeks immediately after the injury. The BBB locomotor rating scale is a motor function evaluation standard widely used in spinal cord injury experiments, and scores the behavior of the hind limbs of the target rat based on the evaluation standard. The detailed evaluation criteria are shown in FIG.

<4-7. Result>
The results of immunohistochemical staining are shown in FIG. As shown in FIG. 13, most of the regenerated axons showing GAP-43 positive infiltrated into GFAP positive cells by the combined use of cell transplantation of S / S: DPC-S group and daily administration of edaravone.
In addition, GFP-positive cells at the site of spinal cord injury when cell transplantation in the S / S: DPC-S group and edaravone administration were used in combination, as compared to the cell transplantation group in the S / S: DPC-S group. Was significantly increased (FIGS. 14a-c). This indicates that even when dental pulp cells not treated with FGF2 are transplanted to the site of nerve injury, the engraftment of dental pulp cells can be significantly increased by the combined use of an active oxygen scavenger.
Furthermore, as shown by the BBB score results in FIG. 15, in the case of the combined use of cell transplantation in the S / S: DPC-S group and edaravone administration, only edaravone administration and S / S: DPC-S Compared with the BBB score of cell transplantation alone (without edaravone administration), it showed a significant improvement effect on motor activity. This result indicates that when dental pulp cells are transplanted to a nerve injury site, the effect of improving motor activity can be improved in addition to the improvement of the engraftment rate of dental pulp cells by using an active oxygen scavenger in combination. ..

Claims (5)

A transplant material for the treatment of nerve damage containing dental pulp cells expressing the GABRB1 gene and having resistance to active oxygen.
A transplant material for treating nerve injury , wherein the dental pulp cells are treated with FGF2 and have an increased expression level of the GABRB1 gene as compared with the dental pulp cells not treated with FGF2 . The therapeutic material for transplantation of nerve injury according to claim 1 .
A therapeutic implant in which the nerve injury is spinal cord injury, cerebral infarction, intracerebral hemorrhage, submucosal hemorrhage, spinal cord hemorrhage, nerve compression injury due to herniated disk, sciatic nerve pain, or peripheral nerve injury due to diabetes. The therapeutic implant according to claim 1 or 2, which is used in combination with an active oxygen scavenger for the treatment of nerve injury. It ’s a way to treat nerve damage.
A therapeutic method comprising the step of transplanting the nerve injury therapeutic transplant material according to any one of claims 1 to 3 to a target nerve injury site other than humans. It is a method of manufacturing a transplant material for the treatment of nerve damage.
A step of culturing the dental pulp cells in a medium containing FGF2 and a step of measuring the expression of the GABRB1 gene in the dental pulp cells.
A production method comprising the step of selecting dental pulp cells expressing the GABRB1 gene.

Images