TWI628280B - Neural crest stem-like cell and use thereof in treatment of neural deficit/injury - Google Patents

Abstract

The present invention provides a neural stem cell which transfects a plastid containing a forkhead box (FOX) D3 gene into an adult cell, and a nerve trunk stem cell for preparing a drug for repairing a damaged nervous system. Use; and such neural stem cells can provide a pharmaceutical composition for repairing damage to the nervous system.

Description

Neural nerve stem cells and their use for preparing drugs for repairing damaged nervous system

The present invention provides a nerve-like spinal stem cell, particularly for use in repairing damage to the nervous system.

Repairing the central nervous system has long been one of the medically insurmountable problems, such as those with neurodegenerative consequences of Alzheimer's disease, Parkinson's disease, and stroke. For these conditions, it is hoped that a cell population that rebuilds the neural network system and restores the function of the nervous system can be developed. For this reason, scientists have continuously developed neural stem cells and progenitor cells. Until now, pluripotent neural progenitor cells have been known to regulate restricted or restricted glial cells in the early differentiation pathway.

Neural stem cells can regenerate from diseased or damaged tissues; however, this treatment will require precise control of the function of the cells to establish the necessary cell types. There is currently no complete understanding of the mechanisms regulating cell proliferation and differentiation, and thus it is difficult to fully exploit the plasticity of neural stem cell populations from any particular region of the brain or developing fetus.

In the central nervous system, regenerative capacity is traditionally considered to be limited, leaving only a limited number of neural stem cells in adulthood. Therefore, the donor brain of the fetal brain is gradually developed, but this method has a huge medical ethical problem in addition to immune rejection, and the ability of the neural stem cells to lose cell division after transplantation, Whether this method is suitable for treating a large number of patients is questioned. In order to overcome the cells given to a specific source of the patient, the use of skin cell trans-differentiation as neural stem cells and/or neurons has been developed, but it has never been confirmed that the cells can be fully differentiated into a fully functional and stable Type of neural precursor cells or neural stem cells. Therefore, there is a stable phenotype after transdifferentiation It has always been one of the major challenges facing the field.

In view of the above problems, there is a need for a stable and effective treatment using cells selected from adult neural stem cells, neural precursor cells, neurons, glial cells, and other types of cells for various neurological disorders and nerves. Treatment of the disease.

In view of the above, it is an object of the present invention to provide a neural stem-derived stem cell cell which transfects a plastid of a forkhead box (FOX) D3 gene into a single adult cell. And get.

It is still another object of the present invention to provide a pharmaceutical composition comprising such neural stem stem cells.

Another object of the present invention is to provide a use of such neural spinal stem cells for the preparation of a medicament for repairing damage to the nervous system.

In one embodiment of the invention, the plastid containing the forkhead box (FOX) D3 gene is transfected into the adult cell line by substrate-mediated gene delivery transfection, And the substrate is a chitosan substrate.

In an embodiment of the invention, wherein the adult cell line is a fibroblast.

In an embodiment of the invention, the neural stem cells of the type enhance the expression of a neural ridge gene, and the neural ridge gene is SOX10 or CD271.

In an embodiment of the invention, wherein the neural stem stem cells increase the expression amount of a stem cell gene, and the stem cell gene line is selected from the group consisting of OCT4, SOX2, and NANOG.

In an embodiment of the invention, the neural stem cells of the type enhance the expression of a nerve-related gene, and the nerve-related gene is selected from the group consisting of Nestin and β-tubulin (β-tubulin). ) and the group of GFAP.

In one embodiment of the invention, the neural stem cells of the type enhance the expression of a neurotrophic factor, wherein the neurotrophic factor is GDNF or NFIX.

In one embodiment of the invention, the neural stem cells of the type increase the amount of expression of the CXCR4 receptor gene and reduce the amount of CXCL12 chemokine expression.

In an embodiment of the invention, the adult cell is homologous to autologous transplantation or xenograft with the source of damage to the nervous system.

The present invention provides a neural stem-derived stem cell which transfects a plastid containing a forkhead box (FOX) D3 gene into an adult cell, and reprograms the cell to a more primitive neuroblast-derived stem cell, which is related to the nerve ridge. Stem cells and nerve-related performance were higher than those of untransfected cells. The present invention also confirmed by experiments that adult cells transfected with the plastid containing the FOXD3 gene have an excellent therapeutic effect in repairing an animal model in which the nervous system is damaged; meanwhile, the neural stem stem cell line of the present invention can be contained. Obtained from the adult cells transfected with the plastid of the FOXD3 gene. Therefore, the neural stem stem cells such as the present invention can be applied to the treatment of repairing damage to the nervous system.

The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims.

Figure 1A is a schematic representation of transfection of plastids into fibroblasts via conventional transfection methods (transfection reagents) and substrate-inducible delivery (no transfection reagent).

The first panel B is a cell type in a chitosan substrate which is cultured in a conventional culture dish (TCPS) and a substrate-induced transfer method (no transfection reagent) via a conventional transfection method (transfection reagent).

Figure 2A shows the transfection efficiency of transfected FOXD3 plastid human fibroblasts by immunofluorescence staining.

Figure B is a flow cytometry to confirm the transfection efficiency of transfected FOXD3 plastid human fibroblasts.

Figure 3A shows the effect of FOXD3 plastid transfection on cell viability.

Figure 3B shows the effect of FOXD3 plastid transfection on cell proliferation rate.

Figure 4A shows the effect of the transfected FOXD3 plastid cells of the present invention on the amount of neuronal gene expression.

Figure 4B is a graph showing the effect of the transfected FOXD3 plastid cells of the present invention on the expression of stem cell-associated genes.

Figure 5 is a diagram showing the transfection of FOXD3 plastid cells of the present invention for nerve correlation The effect of the amount of gene expression.

Figure 5B is a graph showing the results of immunofluorescence staining of nerve-related genes by the transfected FOXD3 plastid cells of the present invention.

The sixth panel shows the effect of the transfected FOXD3 plastid cells of the present invention on the amount of neurotrophic factor expression.

Figure 7A is a schematic diagram of the present invention for injecting FOXD3 plastid cells into embryos.

Figure 7B shows the frequency of spontaneous squatting of zebrafish after injection of FOXD3 plastid cells in the present invention.

Figure 7C shows the frequency of crimp contraction recovery of zebrafish after injection of FOXD3 plastid cells in the present invention.

Fig. 7D is an image diagram showing the position of the cells after injection of the transfected FOXD3 plastid cells into the embryo.

Figure 7 is a diagram showing the embryo hatching rate of the present invention after injection of the FOXD3 plastid cells into the embryo.

Figure 7 is a schematic view of a zebrafish traumatic brain injury caused by a needle in the present invention.

Figure 7G shows the recovery rate of zebrafish traumatic brain injury treated with transfected FOXD3 plastid cells.

The present invention provides a neural stem-derived stem cell which transfects a plastid containing a forkhead box (FOX) D3 gene into an adult cell, and reprograms the cell to a more primitive neuroblast-derived stem cell, which is related to the nerve ridge. Stem cells and nerve-related performance were higher than those of untransfected cells. The present invention compares the difference of cells after transfection by using substrate-mediated gene delivery and traditional transfection methods, and confirms that both transfection methods can successfully convert FOXD3 plastids by immunofluorescence staining. Dyeing into cells; using multi-functional microplate analyzer to analyze the difference in cell proliferation after transfection of two transfection methods, confirming that transfection of FOXD3 plastids contributes to cell proliferation; and then using the reverse reverse transcription polymerase chain reaction ( Real-time RT-PCR analysis of neural ridge genes (including FOXD3, SOX10, and CD271) and stem cell-related genes (including OCT4, SOX2, and NANOG), genes related to neurogenic precursor cells (Nestin, β-tubulin, MAP2, and GFAP), neurotrophic factors (including NFIX, NGF, BDNF, and GDNF) As well as the amount of chemokine/receptor genes (including CXCL12 and CXCR4), it was confirmed that the transfection of FOXD3 plastids upregulated the expression of the above genes. Further, the present invention injects cells transfected with FOXD3 plastids into the zebrafish embryonic nerve defect module to evaluate the effect of nerve regeneration in vivo, and confirms that the transfected FOXD3 plastid cells are damaged in vivo in vivo. The therapeutic effect of the zebrafish experiment.

definition

The experimental data of the present invention are all expressed in mean ± standard deviation. The reproducibility of each experiment in vitro was independently confirmed three times from cells of three different grantors. Statistical differences between experimental group tissues were confirmed by one-way ANOVA, and the experimental results were statistically considered significant when the p value was <0.05.

Example 1 Construction of forkhead box (FOX) D3 plastid

The present invention refers to the method of Pan G, Li J, Zhou Y, Zheng H, Pei DA negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal. FASEB J. 2006; 20 (10): 1730-2, The forkhead box (FOX) D3 is inserted into the Eco RV cutting position of the plastid by the reverse transcription polymerase chain reaction (RT-PCR) FOXD3 sequence and has a Flag tag. The tag is in the pCR3.1 vector.

Example 2 Preparation of Chitosan Substrate

First, a chitosan powder having a molecular weight of 510 kDa (available from Sigma, USA) having a deacetylation degree of 77.7% was detected by Nuclear Magnetic Resonance Spectroscopy (NMR). The chitosan powder was dissolved in 1% acetic acid and gently stirred at room temperature for 12 hours to obtain a 1% chitosan solution. The solution was filtered through a mesh of 100 μm, and 300 μL of the chitosan solution was uniformly coated on a 15 mm slide and placed in a petri dish. After drying, it was immersed in a 0.5 N sodium hydroxide solution for 3 minutes. After soaking, it is rinsed with a large amount of distilled water, and after drying, a chitosan substrate is obtained.

Example 3 Culture of human dermal fibroblasts

The human fibroblast cell line is obtained from an adult foreskin after circumcision surgery. In the present invention, the method involves human tissue, and the present invention complies with the ethical guidelines for human medical research and is approved by the Ethics Review Committee of the Three Military General Hospital of the Republic of China. The present invention utilizes a known method for the separation and proliferation of human fibroblasts. First, the foreskin tissue is decomposed by collagenase, and the cell suspension is centrifuged, followed by containing 10% fetal bovine serum (purchased from Gibco). 1% penicillin/streptomycin (purchased from Invitrogen), Dulbecco's modified Eagle's medium (purchased from Gibco) was resuspended, and the cells were cultured at 37 ° C, 5% CO ₂ In the incubator, when the cells reached 80% confluence, the fibroblasts were subcultured with 0.25% trypsin/EDTA solution (purchased from Gibco), and the culture medium was changed every three days. Fibroblasts from passage 3 to passage 8 are available for subsequent experiments.

Example 4 forkhead box (FOX) D3 transfection

The present invention transfects the FOXD3 sequence into human fibroblasts using a substrate-mediated gene delivery method. First, human fibroblasts were seeded on a chitosan substrate (about 2.8 × 10 ⁴ cells/cm ² ) in a 24-well plate for 12 hours, followed by 1 μg of FOXD3 in 1 mL per well. The plastid solution replaced the medium and the fibroblasts were exposed to the plastid for 24 hours. Thereafter, each well was replaced with fresh medium and cultured for 36 hours, and fibroblasts transfected with FOXD3 plastids were collected by aspirator.

To compare the difference in transfection between the substrate-induced delivery mode and the conventional transfection mode, the present invention transfects fibroblasts using Polyfect transfection reagent (Qiagen) for comparison, using the manufacturer's operating manual. The fibroblasts were cultured in 24-well general culture dishes (TCPS) (about 2.8 × 10 ⁴ cells/cm ² ) for 24 hours, followed by 1 mL of 1 μg of FOXD3 plastid solution and 2.5 μg of Polyfect in each well. The staining reagent replaced the medium and the fibroblasts were exposed to the plastid for 24 hours. Thereafter, each well was replaced with fresh medium and cultured for 48 hours, and the attached cells were suspended with trypsin and the transfected fibroblasts were collected.

Example 5 Analysis of transfection efficiency and cell proliferation rate

The invention utilizes immunofluorescence staining method to evaluate the transfection of FOXD3 plastid fiber matrix Cell effect, and FLAG fusion protein was detected using an anti-FLAG antibody. First, the fibroblasts were fixed in a 4% formaldehyde solution for 10 minutes, permeabilized with 0.1% Triton X-1002 in phosphate buffer (Phosphate Buffered Saline, PBS) for 10 minutes, and then 1% fetal. Bovine serum (BSA) was reacted as a blocking solution for 10 minutes, and stained with FLAG as a primary antibody (purchased from Genetex) at 4 ° C until the next day. The cells were washed with PBS buffer at room temperature and treated with PE Goat Anti-Rabbit IgG secondary antibody (purchased from BioLegend) for 1 hour. The sample was mounted and observed under fluorescent microscopy.

To confirm the cell proliferation rate after FOXD3 transfection, the present invention utilizes DNA Hoechst 33528 fluorescent staining assay (purchased from Sigma) to use a multi-plate microplate reader (fluorescent mode, SpectraMax M5) at 365 nm and 458 nm. The fluorescence intensity was confirmed at the wavelength, and the fluorescence intensity value was used to calculate the number of cells using a standard curve.

5.1 Cell type of transfected FOXD3 plastid human fibroblasts transfected in different ways

Figure A is a schematic diagram of transfection of plastids into fibroblasts via conventional transfection methods (transfection reagents) and substrate-inducible delivery (no transfection reagent); Figure 1 B is a traditional transfection The method of culturing on a general culture dish (TCPS) and the substrate-induced transfer method in the cell type of the chitosan substrate, it can be observed that the cells inoculated on the general culture dish (TCPS) are dispersed, and in a few On the butanose substrate, the cells aggregated into a spherical shape, but the cell type on the general culture dish (TCPS) or the chitosan substrate did not affect the FOXD3 plastid transfection effect.

5.2 FOXD3 plastid transfection effect

To confirm the transfection efficiency of transfected FOXD3 plastid human fibroblasts, the traditional transfection method (transfection reagent) and substrate induction transfer method (no transfection reagent) were used to transfect the plastid into fibroblasts, and then use the immunization. Fluorescent staining confirmed whether the transfection was successful. As shown in Figure A, the fluorescence intensity of the cells using the traditional transfection method (expressed as TCPS+FOXD3) is slightly higher than that of the substrate (indicated by chitosan+FOXD3), while untransfected cells. (represented by TCPS or chitosan) is not recognized by antibodies. Then, flow cytometry was used to analyze the transfection efficiency of FOXD3 transfected into cells. As shown in Figure B, the transfection efficiency of FOXD3 transfected with traditional transfection method (expressed as TCPS+FOXD3) was higher than that of substrate. (Expressed as chitosan + FOXD3), the transfection efficiency was about 32% and about 13%, respectively. Untransfected cells (represented by TCPS or chitosan) Now <2%. The results showed that both transfection methods successfully transfected FOXD3 plastids into cells, and the transfection efficiency was higher in the traditional transfection mode.

5.3 Effect of FOXD3 plastid transfection on cell viability and cell proliferation rate

The effect of conventional transfection (transfection agent) and substrate-induced delivery mode (no transfection agent) on cell viability and cell proliferation after cell transfection. As shown in Figures A and B of the third panel, transfection of plastids (expressed as chitosan + FOXD3) by substrate-induced delivery did not affect cell viability. Conversely, transfection of plastids into cells using a transfection reagent (represented by TCP2+FOXD3) results in partial cell death with a survival rate of approximately 72%; fibroblasts transfected with FOXD3 plastids have a higher cell proliferation rate than Transfected cells (represented by TCPS or chitosan) are fast. The rate of proliferation of cell culture on different substrates is also different. The cell proliferation rate of cells in a general culture dish (expressed by TCPS) is faster than that of chitosan substrate (expressed as chitosan), and transfected with FOXD3 plastids. The cells (represented by TCP2+FOXD3) also proliferated at a rate faster than the chitosan substrate (expressed as chitosan + FOXD3) on TCPS. These results show that transfection of FOXD3 plastids contributes to cell proliferation. In addition, the method of transfection with a substrate does not affect the survival rate of the cells.

Example 6 Gene Expression

The present invention utilizes real-time RT-PCR to analyze neural ridge genes (including FOXD3, SOX10 and CD271), stem cell related genes (including OCT4, SOX2 and NANOG), and related genes of neural precursor cells. (Nestin, β-tubulin, MAP2, and GFAP), neurotrophic factors (including NFIX, NGF, BDNF, and GDNF) and chemokine/receptor genes (including CXCL12 and CXCR4) The amount of performance. Total RNA was extracted from fibroblasts transfected with no FOXD3 plastids and fibroblasts transfected with FOXD3 plastids for 72 hours using Trizol® reagent (purchased from Invitrogen), without FOXD3 plastid transfected fibroblasts Contains two cells cultured on a common culture dish (TCP) and chitosan substrate; fibroblasts transfected with FOXD3 plastid contain traditional means (transfection agent) and substrate-induced delivery mode (no transfection agent) ) both cells, then reverse transcribed RNA first to cDNA and the amplification reaction via RevertAid ^TM first strand cDNA synthesis kit (first strand cDNA synthesis kit) (available from MBI Fermentas Inc.). The DyNAmo Flash SYBR Green Qpcr Kit (available from Finnzymes) was used to perform a reverse reverse transcription polymerase chain reaction with a PTC200 Thermal Cycler (purchased from MJ Research), and the genes were normalized with the GAPDH gene (housekeeping gene). The amount of expression, the pair of primers for each gene is shown in Table 1.

In addition, the present invention analyzes the expression of Nestin and GFAP genes transfected with FOXD3 plastid fibroblasts by immunofluorescence staining. First, the fibroblasts were washed with PBS buffer and fixed in a 4% formaldehyde solution for 10 minutes, permeabilized with PBS buffer containing 0.1% Triton X-1002 for 10 minutes, and then 1% fetal bovine serum ( BSA) was reacted as a blocking solution for 1 hour, and stained with GFAP as a primary antibody (purchased from Chrmicon) at 4 ° C until the next day. The cells were washed with PBS buffer at room temperature and treated with PE Goat Anti-mouset IgG secondary antibody (purchased from Life Technologies) for 1 hour. The sample was mounted and observed under fluorescent microscopy.

6.1 Transfection of FOXD3 plastid cells in neural ridge gene markers and stem cell gene markers

The effect of cells transfected with FOXD3 plastids on neural ridges and stem-related genes. As shown in Figure 4A, the cells transfected with FOXD3 showed higher ridge-related genes (FOXD3 and SOX10) than untransfected cells; Cells transfected in a conventional manner (represented by TCPS+FOXD3) exhibited a higher FOXD3 gene expression than cells transfected with the substrate-inducing mode (expressed as chitosan+FOXD3). The expression levels of the SOX10 gene and the CD271 gene were higher in cells transfected with the substrate-inducing mode (expressed as chitosan+FOXD3). In terms of dryness-related gene expression, as shown in Figure B, the cells germinated on the chitosan substrate (expressed as chitosan) are higher than the cells that are not pelletized on a common dish ( According to TCPS), the cells on the chitosan substrate that were not transfected with FOXD3 plastids (expressed as chitosan) were higher in the OCT4, SOX2 and NANOG genes than in the culture dishes. Cells stained with FOXD3 plastids (represented by TCPS). In addition, the dry-related genes transfected with FOXD3 cells performed better than untransfected Cells, except for the OCT4 gene expression transfected with FOXD3 cells (represented by TCPS+FOXD3) in a conventional manner, were not significantly different from untransfected cells. Cells transfected with FOXD3 (expressed as chitosan+FOXD3) using substrate-induced transfer were significantly more abundant than non-transfected cells (>2.5-fold). All of the above results confirmed that the cells transfected with FOXD3 plastids up-regulated neural ridges and stem-related genes.

6.2 Neuro-related gene expression of transfected FOXD3 plastid cells in neuroblast-like precursor cells

The effect of cells transfected with FOXD3 plastids on the expression of nerve-related genes. As shown in Figure 5A, cells transfected with FOXD3 plastids upregulate Nestin and β-tubulin regardless of any transfection method. (β-tubulin), the amount of GFAP gene expression, the MAP2 gene is the opposite. On the other hand, the substrate-induced delivery of FOXD3 plastid-transfected cells (expressed as chitosan+FOXD3), Nestin, β-tubulin, and GFAP genes were compared. high. As shown in Figure 5B, immunofluorescence staining showed the presence of nestin (Nestin) and GFAP protein in cells transfected with FOXD3 plastids. The above results demonstrate that transfection of FOXD3 plastids into fibroblasts contributes to neurologically relevant performance.

6.3 Performance of neurotrophin and chemokine/receptor genes transfected with FOXD3 plastid cells

The present invention further analyzes the gene expression of neurotrophic factors by transfected FOXD3 plastid cells. As shown in the sixth panel, among all the groups, human fibroblasts (expressed as TCP) which were not transfected in the general culture dish had the highest expression in the NGF and BDNF genes, and the expression level of GDNF was low. Culture of human fibroblasts that were not transfected with chitosan substrates (expressed as chitosan) and transfected in a conventional manner (represented by TCP+FOXD3) with low expression in NGF and BDNF genes and in GDNF High performance. Human fibroblasts (expressed as chitosan+FOXD3) transfected with FOXD3 plastids in the substrate-inducing mode were the highest in GDNF. Cells transfected with FOXD3 plastids showed increased expression in the NFIX gene, whereas cells not transfected with FOXD3 plastids were cultured in a common dish (represented by TCP) or chitosan substrate (expressed as chitosan) The amount of gene expression in NFIX is close. The results showed that transfection of FOXD3 plastids contributed to the expression of neurotrophic factor GDNF.

The effect of fibroblast pelleting and FOXD3 transfection on chemokine/receptor, culturing cells germinated on chitosan substrate compared to cultured in general medium The cells have higher chemokine/receptor gene expression, and the FOXD3 plastid transfected cells showed a significant increase in CXCR4 gene expression, while the CXCL12 chemokine expression was completely opposite to the CXCR4 receptor gene. The results confirmed that FOXD3 transfected cells would move to the injured site, especially the cells that formed on the chitosan substrate.

Therefore, the transfected FOXD3 plastid cells of the present invention can enhance the neural ridge genes (including FOXD3, SOX10 and CD271), stem cell related genes (including OCT4, SOX2 and NANOG), and related genes of neural precursor cells (Nestin). , β-tubulin and GFAP), neurotrophic factors (including NFIX and GDNF), and the amount of expression of the receptor gene CXCR4; and decreased expression of the chemokine CXCL12. Therefore, the transfected FOXD3 plastid cells of the present invention can express the characteristics of the neural stem cells, and the present invention further confirms the therapeutic effect of the transfected FOXD3 plastid cells by the in vivo experiment of the nerve damaged zebrafish.

Example 7 Evaluation of Neurological Repair of Transfected FOXD3 Somatic Cells in Vitro

The present invention injects neural stem cells transfected with FOXD3 plastids into the zebrafish embryonic nerve defect module to evaluate the efficacy of nerve regeneration in vivo, and utilizes coiling contraction (indicator of motor function) and Embryo hatchability (CNS functional palliative) evaluates the repair of neurological deficits through various treatments. All procedures are ethical and approved by the Institutional Animal Care and Use Committee (IACUC).

7.1 Coiling contraction analysis of zebrafish injected with transfected cells

The zebrafish is divided into six groups according to the present invention. The first group is a zebrafish embryo cultured in a normal medium as a blank group (indicated by WT); the other group is as shown in the seventh panel A, and E3 containing 2% ethanol (EtOH). The embryo culture medium (containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl ₂ and 0.33 mM MgSO ₄ ) was cultured for 4 hours to facilitate the development of the nervous system. The zebrafish was used as an experimental group, and then replaced with a normal medium, and then various embryos were subjected. The treatment comprises the second group of human fibroblasts (about 3×10 ² cells) cultured in a general medium to the embryos (represented as dispersed cells), and the third group is human fibers cultured in the chitosan by injection. Mother cells (approximately 3 × 10 ² cells) to embryos (expressed as spheroid cells), and a fourth group of fibroblasts (approximately 3 × 10 ² cells) transfected with FOXD3 plastids in a conventional manner to embryos Fibroblasts (about 3 × 10 ² cell number) transfected with FOXD3 plastids into embryos (expressed as blast cells + FOXD3) were injected into the FOXD3 plastids by substrate-inducing transfer in the fifth group. In addition, the sixth group was treated with ethanol only as an untreated control group (expressed as EtOH).

The embryos and seedlings were observed by a dissecting microscope of the present invention, and the movement of the embryos was recorded by a CCD camera, and analyzed in the 18 Somite stage, a side-to-side analysis by ImageJ software. For the coiling contraction experiment, the total number of contractions in 2 minutes was calculated after 24 hours of fertilization (hpf) to show the number of contractions per second (Hz), and the hatching rate of the embryos was evaluated 48 hours after fertilization (hpf). This value showed that the zebrafish of the experimental group recovered neurological function compared with the blank group and the control group.

Evaluation of functional recovery of neural stem stem cells, such as different transfection methods, injected into nerve-damaged zebrafish. The frequency of the tail of the zebrafish was observed 24 hours after the embryo was fertilized. As shown in the seventh panel B, the spontaneous appendix was observed at the 18-segment stage after the cell injection, and it was found that the untransfected cells were injected. Cells or scattered cells are expressed in zebrafish of nerve-damaged embryos, only about 25% of which are slightly spontaneous spontaneous appendix, and the remaining 75% are unreacted, and the group of untreated cells (expressed by EtOH) No difference. About 25% of the zebrafish transfected with the transfected FOXD3 plastids transfected with the traditional transfection method showed normal spontaneous appendix (expressed as scattered cells + FOXD3); transfected FOXD3 plastids by substrate-induced transfer About 40% of the zebrafish of the globular cells have a normal expression of spontaneous appendix (expressed by spheroid cells + FOXD3). Cells transfected with FOXD3 plastids had higher frequency of appendix than untransfected cells. The zebrafish transfected with FOXD3 plastids in the induction-transfer mode of the substrate had the highest frequency of appendix, followed by the zebrafish transfected with the transfected FOXD3 plastids transfected with the traditional transfection method, but not The zebrafish of the transfected cells did not differ from the group of untreated cells either in the cells cultured on the chitosan substrate or in the single cells cultured in the general culture dish.

Results of coiling contraction 24 hours after embryo fertilization, as shown in Figure 7C, zebrafish transfected into FOXD3 plastid blast cells (represented by blast cells + FOXD3) by injection-inducing transfer The highest curl contraction recovery frequency is >0.06 Hz, followed by zebrafish transfected with FOXD3 plastids transfected with traditional transfection (expressed cells + FOXD3), the worst is injection untransfected Cell of zebrafish. This result confirmed that the frequency of curl contraction recovery was correlated with early spontaneous contraction.

7.2 Tracking of transfected cells to embryos

The present invention performs transfection cell tracking, and the fibroblasts in vitro are labeled with a red fluorescent cell tracker PKH26 (purchased from Sigma-Aldrich), and the cells are suspended from the cell culture flask by trypsin. It was labeled with PKH26 (0.5 mL 1 × 10 ³ M for 1 × 10 ⁶ cell number), after which the cell culture step was the same as above. The hatching rate of the zebrafish embryos was observed under a fluorescent microscope 48 hours after fertilization (hpf).

The cells were injected into the nerve-damaged zebrafish embryos and the cell position was observed after the embryos hatched. As shown in Figure 7D, it was found that cells that were not transfected with FOXD3 (represented by dispersed cells or spheroid cells) were almost all in the yolk. Cells transfected with FOXD3 plastids (expressed as dispersed cells + FOXD3 or spheroid cells + FOXD3) are transfected into the nucleated cells of FOXD3 plastids in the brain region and induced by substrate induction. The number of cells + FOXD3 is greater than the number of dispersed cells (expressed as dispersed cells + FOXD3) transfected by conventional transfection. These results demonstrate that cells transfected with FOXD3 can restore damaged nerves, especially in cells that are induced by substrate-induced delivery.

The embryo hatching rate of zebrafish was observed 48 hours after embryo fertilization. As shown in Figure 7E, the embryos (spheroid cells + FOXD3) which were transfected with FOXD3 plastids by substrate-induced transfer were the highest. Embryo hatching rate is about 68%. Embryo hatching rate of embryos transfected with FOXD3 plastids (expressed cells + FOXD3) transfected with traditional transfection method is about 55%, untransfected cell group ( The spheroid cells or scattered cells were not significantly different from the untreated cells (expressed as EtOH), and the embryo hatching rates were embryos that were not transfected into cells that were germinated on chitosan substrates, respectively. The hatching rate is about 26%, which is an unimmunized embryo that is cultured on a chitosan substrate into a sphere (expressed by a dispersed cell). The hatching rate is about 25%, and the embryo of the untreated cell (in terms of globular cells) EtOH indicates that the hatching rate is about 21%.

7.3 Efficacy of injection of transfected cells in a zebrafish traumatic brain injury module

To further confirm the therapeutic effect, the present invention further applies cells transfected with FOXD3 plastids to an adult zebrafish module for treating traumatic brain injury. Adult zebrafish were maintained at 28 ° C for 14 hours light and 10 hours dark. All adult zebrafish used in the present invention are 2.8 to 3.8 cm in length, and adult zebrafish are divided into five groups (each group, n=5). The first group was the adult zebrafish without damage as a blank group; the other groups, as shown in Figure F, showed a cerebellar injury with a 27G needle (purchased from Thermo Scientific), which was transfected with FOXD3 plastids, respectively. Cells and non-transfected cells coated with chitosan-based self-healing hydrogel, wherein chitosan-based self-healing hydrogels are composed of A mixture of telechelic difunctional poly(ethylene glycol (DF-PEG) solution and ethylene glycol chitosan, the rest without any treatment as an untreated control group. Before the zebrafish sacrifice The zebrafish exercise analysis was carried out for 8 days, swimming in a container of 260 mm × 90 mm × 50 mm, and the recovery function (%) was used to evaluate the motor function of the zebrafish (the untreated control group (or treatment group) swimming rate/blank The swimming rate of the group × 100%) was analyzed by Image J software.

Adult zebrafish modules with traumatic brain injury may not swim or swim imbalance during swimming. Assessment of functional recovery of adult zebrafish swimming rates after various treatments. As a result, as shown in the seventh panel G, the glomerular cells transfected with FOXD3 plastids (represented by blast cells + FOXD3) after 8 days of substrate induction showed the highest recovery rate (62%), followed by the highest recovery rate (62%). Treatment with transfected cells transfected with conventional transfection (expressed in cells + FOXD3) showed a 50% recovery rate, and untransfected cell therapy (expressed in scattered cells) showed a 25% recovery rate, untreated control The group showed a recovery rate of 20% (expressed as untreated). These results confirm that cells transfected with FOXD3 plastids have the ability to repair central nervous system damage in vivo.

In summary, the present invention provides a neural stem-derived stem cell which transfects a plastid containing a forkhead box (FOX) D3 gene into an adult cell, and reprograms the cell to a more primitive neural stem cell. The expression of nerve ridges, stem cells, and nerve-related performance are higher than those of untransfected cells, especially those transfected by the inducing transfer of raw substrates. The fibroblasts transfected with FOXD3 plastids were applied to nerve-damaged zebrafish, and it was found that the nerve function recovery effect (about 62% recovery rate) of cells injected with FOXD3 plastids was better than that of traditional transformation. The dye was transfected into the cells of FOXD3 plastid (about 50% recovery rate), while the cells not transfected with FOXD3 plastid had no significant effect on nerve repair, so transfected somatic cells containing the plastid of FOXD3 gene for repairing the nervous system In the damaged animal model, there is an excellent therapeutic effect. Meanwhile, the neural stem stem cells such as the present invention are not cytotoxic and safe. Therefore, the neural stem stem cells such as the present invention can replace the dilemma of insufficient source of nerve-related cells, and thus can be applied to Many diseases are treated.

<110> National Taiwan University

<120> Neural nerve stem cells and their use for preparing a drug for repairing damage to the nervous system

<130> 104B0453-I1

<160> 44

<170> PatentIn version 3.5

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<223> Synthetic primer

<400> 5

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

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<400> 6

<210> 7

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<212> DNA

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<212> DNA

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Claims (8)

Use of a neural stem-like stem cell for preparing a drug for repairing a damaged nervous system, wherein the neural stem cell line is transfected into a fiber matrix by a plastid containing a forkhead box (FOX) D3 gene Obtained from cells. The use according to claim 1, wherein the plastid containing the forkhead box (FOX) D3 gene is transfected into the fibroblast cell line using a substrate-mediated gene delivery method. Transfected, and the substrate is a chitosan substrate. The use according to claim 1, wherein the neural stem stem cell line enhances the expression of a neural ridge gene, wherein the neural ridge gene system is SOX10 or CD271. The use of claim 1, wherein the neural stem stem cell line enhances the expression of a stem cell gene selected from the group consisting of SOX2 and NANOG. The use according to claim 1, wherein the neural stem stem cell line enhances the expression of a nerve-related gene selected from the group consisting of Nestin and β-tubulin ( --tubulin) and a group consisting of GFAP. The use according to claim 1, wherein the neural stem stem cell line enhances the expression of a neurotrophic factor, wherein the neurotrophic factor is GDNF or NFIX. The use according to claim 1, wherein the neural stem stem cell line enhances the expression amount of the CXCR4 receptor gene and reduces the expression amount of the CXCL12 chemokine. The use of claim 1, wherein the fibroblast and the source of damage to the nervous system are the same autologous or xenograft.