KR101559884B1 - Composition for adhesion or proliferation of dental pulp cells comprising recombinant dentin phosphoprotein - Google Patents

Abstract

The present invention relates to a composition for promoting adherence or proliferation of dental pulp cells containing dentin phosphoprotein as an active ingredient, and a method for improving the adhesion or proliferation of dental pulp cells, Or a method of inducing proliferation. By using the recombinant human dentin phosphate protein capable of mass production using the expression vector of the present invention, it is possible to promote the proliferation of the dendritic cells, increase the cell adhesion capacity, and promote the differentiation into the dendritic cells from the dendritic cells Respectively. Therefore, the recombinant human dentin phosphate protein of the present invention is widely used for promoting the proliferation and adhesion of dendritic cells in the fields of tissue engineering materials, dental bone regeneration therapeutic agents and medicament therapeutic agents related to recovery and regeneration of teeth and bone tissues It will be possible.

Description

Composition for adhesion or proliferation of dental pulp cells comprising recombinant dentin phosphoprotein}

The present invention is a composition for promoting adhesion or proliferation of pulp cells containing dentin phosphoprotein as an active ingredient, and adhesion of pulp cells comprising the step of treating the dentin phosphate protein to pulp cells Or to a method of inducing proliferation.

The tooth has enamel in the outermost layer and hard tissue called dentin in the inner layer, and it is an organ with dentin-producing dentin inside the dentin, and pulp cells in the center. Teeth are formed by induction of developmental processes in the prenatal period, and are functional units constructed by several cell tumors, like organs or organs. Therefore, teeth are not generated by stem cell systems that are generated as various cell types from stem cells such as hematopoietic stem cells or mesenchymal stem cells in adults.

As described above, teeth are tissues that are continuously exposed and used in daily life, such as human food intake or language life, whereas teeth are not spontaneously regenerated in adults. It is an organ that can be easily lost. Accordingly, in recent years, depending on the characteristics of tissues that are not spontaneously regenerated, there are many cases of supplementing them with methods such as dentures or implants. However, the presence or absence of teeth greatly affects the appearance and taste of food, and interest in the development of a tooth tissue regeneration technology for maintaining health and high quality life is increasing.

Recently, a number of studies on a method of regenerating tooth tissue using isolated dentinal cells, etc. are being conducted. In J.Dent.Res., 2002, Vo1.81(10), pp.695-700, in the tooth embryo. It was shown that tooth-like tissues were regenerated by transplanting isolated cells such as epithelial cells or mesenchymal dental cyst cells together with a bioabsorbable carrier into the abdominal cavity of a rat. In addition, Japanese Unexamined Patent Application Publication No. 2004-357567 discloses that dentinal cells isolated from a living body can be cultured with a carrier containing fibrin to form teeth. In addition, several technologies have been invented for the technology of forming teeth in dentinal cells (International Publication No. 2005/014070, International Publication No. 2006/129672).

On the other hand, dentin sialophosphoprotein (DSPP) is a representative dentin-specific protein belonging to a non-collagen protein, and from one gene, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) ) To synthesize two proteins. DSPP, known as an oblast-specific gene, is known to play an important role in the mineralization process of dentin, unlike osteoblasts of osteoblasts, but there is still controversy as to whether it is a factor that regulates or induces differentiation of pulp cells.

Dentin phosphate protein (DPP) refers to a carboxy terminal portion produced by cutting the dentin sialophosphoric acid protein (DSPP). Regarding DPP regarding dental tissue, treatment with mesenchymal cells or osteoblastic progenitor cell lines promotes differentiation into dental blasts (Jadlowiec J et al., J Biol Chem. 2004 Dec 17;279(51):53323 -30) and human pulp cell migration and dentin formation (Yasuda Y et al., J Endod. 2008 May;34(5):575-8). However, compared to DSPP, studies related to dental tissue regeneration for DPP are still insufficient.

Under this background, the present inventors have produced and isolated DPP, which is easy to produce, isolate, and purify with a smaller amino acid sequence compared to DSPP, using a recombinant expression vector, so that DPP promotes adhesion, proliferation and differentiation of pulp cells. The present invention was completed by finding out.

One object of the present invention is to provide a composition for promoting adhesion or proliferation of dental pulp cells comprising dentin phosphoprotein as an active ingredient.

Another object of the present invention is to provide a method for inducing adhesion or proliferation of pulp cells, comprising the step of treating pulp cells with dentin phosphate protein.

As one aspect for achieving the above object, the present invention provides a composition for promoting adhesion or proliferation of dental pulp cells comprising dentin phosphoprotein as an active ingredient.

The term "dentin" of the present invention is a calcified tissue containing collagen from the external mesenchymal origin, and is a white hard tissue constituting most of the teeth. It is also called hemorrhoids, and it is covered with enamel at the crown and cement at the root, so it is not exposed to the surface of the tooth.However, as the enamel wears out with age, dentin is exposed at the tip or occlusal surface of the crown. Dentin is a kind of bone tissue, but unlike normal bones, the body of the cells that make dentin exist in the pulp, and only the protrusions extend into the dentin.

The term "dentin phosphoprotein" of the present invention is a protein found in tooth tissue, and refers to a fragment containing a carboxyl terminal portion when the dentin sialophosphoprotein is cut, and the amino terminal portion The fragment containing the is called dentin sialoprotein (dentin sialoprotein).

In the present invention, the dentin phosphate protein is not limited to origin, but is preferably a human-derived dentin phosphate protein, more preferably a dentin phosphate protein having the amino acid sequence of SEQ ID NO: 3.

The term "dental pulp" of the present invention is a soft connective tissue filling the pulp cavity inside a tooth, and is rich in nerves and blood vessels. The pulp nerves form a plexus in the dentin cells in contact with the inner surface of the dentin at the outermost layer of the pulp, and its branches reach the surface layer of the dentin. The "dental pulp cells" of the present invention having the above dimensions are differentiated into odontoblasts forming dentin through a differentiation process to form dentin. The origin is not limited, but may preferably be a human pulp cell.

The term "odontoblast" of the present invention exists on the surface of the cured head in contact with the inner enamel cells of the dentin, and the dentin is made by the dentin by the dentin. In the present invention, the blast cells may include odontoblast-like cells as well as blast cells. The pseudo-denoblasts are cells originally induced to differentiate from stem cells or progenitor cells into dentinal blasts, and refer to cells having similar traits to dentate blasts.

The term "attachment" of the present invention refers to the adhesion of cells, and includes not only the adhesion between cells, but also the phenomenon that the cells adhere to the substrate. In an embodiment of the present invention, in order to confirm the effect of the dentin phosphate protein on the adhesion ability of cells, the degree of adhesion of pulp cells to a plate coated with dentin phosphate protein for a certain period of time was determined by a crystal violet experiment (crystal violet). assay), and it was confirmed that dentin phosphate protein promotes adhesion of pulp cells (Example 4). When the adhesion ability improves, the cells adhere well to the site, and the desired structure can be easily and evenly attached to a certain type of substrate.

The composition of the present invention may be used to attach pulp cells to a specific substrate or site, and may be pre-coated on a specific substrate or site or treated with pulp cells.

The term "proliferation" of the present invention means that the number of cells increases by maintaining the same properties or dividing while differentiating. In the present invention, the proliferation may preferably be proliferation of dental pulp cells, more preferably proliferation of human dental pulp cells (hDPC). In one embodiment of the present invention, in order to confirm the effect of the dentin phosphate protein on the proliferation of pulp cells, the dentin phosphate protein was treated on pulp cells and then confirmed by MTT experiment (Example 5 and FIG. 3). It was confirmed that the proliferation of pulp cells was promoted when the dentin phosphate protein was treated.

Accordingly, the composition of the present invention can be used to promote the proliferation of pulp cells, thereby obtaining a large amount of pulp cells of the same properties.

In an embodiment of the present invention, in order to confirm the effect of dentin phosphate protein on the differentiation of pulp cells into dentin cells, dentin phosphate protein is treated on pulp cells, and ALP (alkaline phosphatase) activity measurement and Alizarin Red S staining are performed. The degree of mineralization was measured.

First, as a result of confirming the ALP activity, there was no difference between the control group that was not treated with anything and the experimental group treated with rhDPP on day 0 of ALP activity, but the experimental group treated with rhDPP afterwards was about 2.3 times and 14 days compared to the control group. The second showed an increase in ALP activity of about 6 times (FIG. 4).

In addition, as the pulp cells differentiate into dentinal cells, calcium is deposited. The Alizarin Red S reagent is a reagent for staining calcium, and the degree of differentiation into dentinal cells was confirmed through the degree of staining with the reagent. When the dentin phosphate protein was treated, there were no cells to be stained on day 0, but it was confirmed that the degree of staining was significantly increased on days 7 and 14 (Examples 6 and 5).

In addition, in an embodiment of the present invention, the expression levels of Col1 (collagenase 1), ALP (alkaline phosphatase specific activity), and DMP-1 (domain matrix protein-1), which are dentinal cell-specific markers for confirming differentiation, are determined by the mRNA level. Reverse transcription PCR and cDNA obtained through it were measured through quantitative real-time PCR (Examples 7, 8 and 6). Through this, when the dentin phosphate protein was treated, DMP-1 did not show any difference, but it was confirmed that the expression of Col1 and ALP was significantly increased.

That is, the dentin phosphate protein of the present invention can promote adhesion of pulp cells, proliferation, or differentiation into dentin cells.

The dentin phosphate protein of the present invention may be a recombinant protein. Dentin sialophosphoprotein (DSPP), previously known as a protein related to dentin differentiation, has a large size and has difficulty in expression, separation, and purification. Accordingly, in the present invention, DNA of dentin phosphate protein, which has a function of promoting adhesion of pulp cells, proliferation and differentiation into dentinal cells, has a short sequence, easy separation and purification, and increases yield, was extracted and introduced into an expression vector. The expression vector can be transduced into a host cell to produce a recombinant protein.

The composition may be used to promote adhesion or proliferation of pulp cells in vitro, or may be used to promote adhesion or proliferation of pulp cells in vivo.

In another aspect, the present invention provides a method for inducing adhesion or proliferation of pulp cells, comprising the step of treating the dentin phosphate protein to pulp cells.

The "treatment of dentin phosphate protein on pulp cells" of the present invention includes, without limitation, direct or indirect methods, in vitro and in vivo methods, and the like. For example, there may be an in vitro treatment method that treats isolated pulp cells in vitro, and an in vivo treatment method such as direct injection or application to tooth tissue in vivo. In one embodiment of the present invention, it was performed by treating the isolated pulp cells in vitro with dentin phosphate protein, and through this, it was confirmed that the dentin phosphate protein promotes adhesion, proliferation and differentiation of pulp cells.

In the present invention, the dentin phosphate protein treated on pulp cells may be prepared including the step of expressing a recombinant human dentin phosphate protein in a host cell transduced with an expression vector comprising the amino acid sequence of SEQ ID NO: 3.

The term "expression vector" of the present invention refers to a DNA or RNA vector capable of transforming a host cell and having an effect of expressing a specific polynucleotide. Preferably, the expression vector means that it is capable of replicating in a host cell. Expression vectors can be prokaryotic or eukaryotic cells, and typically can be viruses or plasmids. The expression vector of the present invention functions in the host cell of the present invention, for example, in cells including bacteria, fungal, endoparasite, arthropod, animal and plant cells (i.e., gene expression Oriented) all vectors. The vector of the present invention can also be used to produce a polypeptide in a cell-free expression system known in the art. In the present invention, the expression vector may preferably be a pBAD/His-A vector having a cleavage map shown in FIG. 1.

The term "host cell" of the present invention refers to a cell that can be transformed with the exogenous polypeptide of the present invention, and may also include transformed recombinant cells. It refers to a cell containing transcription factors, translation factors, or post translational factors so that the introduced expression vector can be properly expressed. Host cells include all cells that can be transformed with the expression vector of the present invention. The host cell of the present invention can endogenously produce the protein of the present invention or can produce the protein after being transformed with the expression vector of the present invention. The host cell of the present invention may be any cell capable of producing the protein of the present invention, and includes bacteria, fungi (including yeast), parasites, nematodes, arthropods, animal and plant cells. As examples of the host cell, Salmonella (Salmonella), E. coli (Escherichia), Bacillus (Bacillus), Listeria monocytogenes (Listeria), saccharose in my process (Saccharomyces), sports help TB (Spodoptera), mycobacteria (Mycobacteria), tricot flu cyano ( trichoplusia ), BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (eg, COS-7) cells, and Vero cells. In addition, as a host cell, for example, E. coli including E coli K12 derivatives; Salmonella (salmonella typhi ); Salmonella typhimurium containing an attenuated strain ( salmonella typhimurium ); Spodoptera Frugi Verda (spodoptera frugiperda ); Trichoplusiani ; And non-tumorigenic mouse myoblast G8 cells (eg, ATCC CRL 1246). In the present invention, the host cell may preferably be a TOP10 cell, which is a kind of E. coli. "Recombinant cells" into which the exogenous polypeptide has been introduced into the host cell includes their direct progeny cells or indirect progeny cells comprising the introduced polynucleotide.

In the present invention, transformation of an exogenous polynucleotide such as the expression vector into a host cell can be performed by any method capable of inserting the polynucleotide into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and fusion of protoplasts. The transformed polynucleotides of the present invention may remain extrachromosomes or may be inserted at one or more positions within the chromosome of the transformed cell in such a way that their ability to be expressed is maintained.

The term "recombinant" of the present invention refers to an act of applying artificial manipulation such as inserting, replacing, or removing a specific natural or mutant gene. In the present invention, "recombinant human dentin phosphate protein" refers to a human dentin phosphate protein in a vector. It refers to a protein product produced by introducing and expressing an expression vector prepared by inserting it into a host cell.

In an embodiment of the present invention, in order to construct an expression plasmid expressing recombinant human dentin phosphoprotein (rhDPP), first, a gene expressing human dentin phosphate protein was amplified through PCR (Polymerase chain reaction). . The amplified PCR product obtained through this was treated with a restriction enzyme and introduced into the pBAD-HisA vector, thereby finally preparing a pBAD-HisA-DPP recombinant vector.

In the present invention, the "dinal cell" includes, without limitation, animal-derived pulp cells, and may include mammalian-derived cells, algae-derived cells, amphibians-derived cells, fish-derived cells, and the like. Preferably, it may be a mammalian-derived pulp cell, more preferably a human pulp cell. Pulp cells are as described above.

Using the recombinant human dentin phosphate protein capable of mass production using the expression vector of the present invention, it is possible to promote the proliferation of pulp cells, increase cell adhesion, and promote differentiation from pulp cells to dentinal cells. Confirmed. Therefore, the recombinant human dentin phosphate protein of the present invention will be widely used to promote the proliferation and adhesion of pulp cells in the development of tissue engineering materials, dental bone regeneration therapeutics, and pharmaceutical therapeutics related to the recovery and regeneration of tooth and bone tissue. I will be able to.

1 is a diagram on the left showing the expression vector pBAD-HisA-DPP, and the diagram on the right is a diagram confirming rhDPP obtained through affinity purification using Ni-NTA resin through coomassie blue staining.
Figure 2 is a view confirming the effect on the cell adhesion activity of hDPC by treatment with rhDPP by a crystal violet assay (crystal violet assay).
3 is a view confirming the effect on the cell proliferation of hDPC by treatment with rhDPP by MTT experiment.
4 is a graph measuring ALP activity in order to evaluate the effect of rhDPP to promote oblast differentiation.
FIG. 5 is a diagram of Alizarin Red S staining for measuring the degree of mineralization in order to evaluate the effect of rhDPP to promote oblast differentiation.
6 is a quantitative real-time PCR (quantitative real-time PCR) of the mRNA expression levels of Col1, ALP, and DMP-1, which are photochemical markers for oblast differentiation, in order to measure the effect of rhDPP on the differentiation of hDPC into oblastoma cells. This is a diagram confirming the results measured through.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1: recombinant dentin Phosphate protein ( rhDPP ) Expression plasmid construction

To construct an expression plasmid expressing recombinant human dentin phosphoprotein (rhDPP), first, a gene expressing human dentin phosphate was amplified through PCR (polymerase chain reaction). PCR was carried out using 50 mM KCl, 10 mM Tris-Cl (pH 8.3), 1.5 mM MgCl ₂ , 0.2 mM dNTPs, 1.25 U of Taq polymerase (ELPis, Daejeon, Korea), and 50 pmol of the following forward primer (SEQ ID NO: 1 ) And a reverse primer (SEQ ID NO: 2).

hDPP forward primer (SEQ ID NO: 1): 5'-TCTCGAGGATGATCCCAATAGC-3'

hDPP reverse primer (SEQ ID NO: 2): 5'-TGGTACCATTAGTCTGATGTGCTATC-3'

As PCR conditions, a cycle consisting of an annealing step at 55°C for 1 minute, an elongation step at 72°C for 1 minute, and a denaturation step at 94°C for 1 minute was repeated 30 times. The amplified PCR product obtained through the above was treated with restriction enzymes Xho I and Kpn I. After the restriction enzyme treatment, the PCR product was ligated to the pBAD-HisA vector to prepare a pBAD-HisA-DPP recombinant vector (Fig. 1A).

Example 2: recombinant dentin Phosphate protein ( rhDPP ) Production and refining

TOP10 cells into which the pBAD-HisA-DPP recombinant vector prepared in Example 1 was introduced were grown overnight in LB (Luria broth)-Amp medium at 37°C. When the concentration of the medium (A ₆₀₀ ) reached 0.6, 0.1% (w/v) L-arabinose was added to induce DPP expression. After incubation at 20° C. for 6 hours, it was deposited through a centrifuge, which was lysed and sonicated. The pulverized product was centrifuged at 14,000 g for 30 minutes in refrigerator (4° C.), and the supernatant was transferred to a new tube.

The crude protein obtained from the sonicated bacterial supernatant is a Ni-NTA resin column (nickel-nitrilotriacetic acid resin column) that binds to the hexahistidine tag present at the amino terminal of the protein; Invitrogen, Carlsbad, CA, USA ) Was purified according to the manufacturer's protocol.

The crude protein and the purified protein were separated using 15% SDS-PAGE (SDS-discontinuous polyacrylamide gel electrophoresis). The purity of the purified protein was confirmed through coomassie brilliant blue staining (FIG. 1).

As can be seen in FIG. 1, rhDPP was obtained with relatively high purity through affinity purification using Ni-NTA resin. When induction with L-arabinose, E. coli TOP10 produced a protein having a molecular weight of 12 kDa, which corresponds to the expected molecular weight of the fusion protein consisting of an amino terminal histidine tag and rhDPP (Fig. 1B). .

Example 3: human pulp cells ( hDPC ) Of separation

Human pulp cells (hDPC) were obtained from the third molar tooth with the written consent of an adult patient aged 19 to 25 years old at the Dental and Craniofacial Clinic at Dankook University College of Dentistry. Specifically, pulp tissue was separated and pulverized from the crown and root, and a solution containing 3 mg/ml of type 1 collagenase and 4 mg/ml of dispase was treated at 37° C. for one hour. The treatment solution was passed through a 70 μm filter to obtain a single cell suspension.

hDPC is heat-inactivated fetal bovine serum (FBS; WELGENE, Daegu, Korea), penicillin G sodium 100 U/ml, streptomycin sulfate 10 μg/ml, and amphotericin B (amphotericin B, WELGENE, Daegu, Korea) in a-minimum essential medium (a-minimum essential medium, a-MEM, WELGENE, Daegu, Korea) containing 25 ㎍ / ml was cultured at 37 ℃ 5% CO ₂ environment. The cultured cells were separated and subcultured after 5 minutes treatment with 0.25% trypsin-EDTA (WELGENE, Daegu, Korea). For cell proliferation and differentiation experiments, hDPCs cultured at passage 3-4 were used.

Example 4: pulp cell adhesion experiment

First, a 24-well plate was coated with rhDPP obtained in Example 2 at 4° C. overnight. In this way, a 24-well plate coated with rhDPP was blocked with PBS (phosphate-buffered saline) containing 1% (w/v) BSA (bovine serum albumin) for 30 minutes, and hDPC at ^{a concentration of 5x10 4} cells/well. Inoculated and washed with PBS. After standing at 37°C for 50 minutes, the plate was washed twice with PBS, and the attached cells were fixed with 3% paraformaldehyde (paraformaldehyde, Sigma, St. Louis, MO, USA) and 2% (v /v) stained with a 5% (w/v) crystal violet solution dissolved in an ethanol/water solvent. After washing with distilled water, the plate was dried. _{The degree of staining was measured with A 570} using a microplate reader (BioRad Laboratories, CA, USA), and a case coated with 1% BSA was used as a control.

As a result, as shown in FIG. 2, the cell adhesion activity of hDPC was significantly increased in a concentration-dependent manner when treated with rhDPP compared to the control (p <0.05). When rhDPP was treated, the cell adhesion activity of hDPC was dramatically increased by 3 times or more at all concentrations compared to the control group. These results show that rhDPP functions to promote the adhesion activity of hDPC. Based on these results, further experiments were carried out by treatment with 5 μg/ml of rhDPP.

Example 5: pulp cell proliferation experiment

Pulp cell proliferation activity was measured through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole, AMRESCO Inc., Solon, OH, USA) experiment, which was manufactured by the manufacturer. Was to measure the number of living cells according to the instructions of. Human pulp cells (hDPC) were cultured for 5 or 10 days at a concentration of ^{1×10 3 cells/well in 24-well plates.} Thereafter, 500 µl MTT solution (5 mg/ml, PBS) was treated in each well and allowed to stand for 4 hours, and then the medium was removed, and a fromazan crystal formed in the cells was dissolved in 200 µl of DMSO. The absorbance of each well at a wavelength of 540 nm was measured using a microplate reader.

When treated with rhDPP, it was confirmed that the proliferation of hDPC was remarkably promoted compared to the control group (FIG. 3). As can be seen in Figure 3, rhDPP increased the cell proliferative activity of hDPC on the 5th and 10th days (p <0.001).

Example 6: Pulp cell differentiation experiment

In order to evaluate the effect of rhDPP to promote the differentiation of dentinal blasts, ALP activity, which is a characteristic of dentinal blasts, was measured, and an Alizarin Red S staining experiment was performed to measure the mineralization activity. First, hDPC was cultured for 0, 7 and 14 days at a concentration of ^{1×10 3} cells/well in a differentiation inducing medium with or without rhDPP. The differentiation induction medium contains 100 μmol/l/ml ascorbic acid, 2 mmol/l b-glycerophosphate, and 10 nmol/l dexamethasone.

Thereafter, hDPC was washed with PBS to measure ALP activity, and 4'C with a lysis buffer containing 1.5M Tris-HCl (pH 10.2), 1 mM ZnCl2, 1 mM MgCl2, and 1% TritonX-100, It was dissolved for 10 minutes. After centrifugation, the ALP activity of the cell lysate was measured using an alkaline phosphate assay kit (Sigma, St. Louis, MO, USA). Absorbance was measured at 405 nm. ALP activity was normalized compared to the control.

In addition, in order to confirm the mineralization activity, hDPC was washed with PBS and fixed in 95% methanol for 30 minutes. Next, the cells were stained with Alizarin Red S solution overnight, washed three times with PBS, and incubated for 15 minutes to remove non-specific staining. The stained patterns on the 0th, 7th and 14th days were confirmed by pictures.

As a result of confirming the ALP activity, there was no difference between the control group and the experimental group treated with rhDPP on day 0 of ALP activity, but the experimental group treated with rhDPP was about 2.3 times higher on day 7 and about 6 times on day 14 compared to the control group. It showed an increase in ALP activity (Fig. 4).

As a result of confirming the mineralization activity of the experimental group treated with rhDPP in hDPC, cells stained with Alizarin Red S were not observed on day 0, and stained cells were observed on days 7 and 14. In particular, it was confirmed that almost all cells were stained on the 14th day (FIG. 5). These results are consistent with the ALP activity results.

Example 7: pulp cell RNA Extraction and cDNA synthesis

Total RNA was extracted according to the manufacturer's instructions using an Easy-spin RNA Extraction kit (iNtRON, Seoul, Korea). The purity of RNA was evaluated by measuring absorbance at wavelengths of 260 nm and 280 nm, and it was considered acceptable when the ratio of _{A 260} /A _{280 was 1.9 to 2.1.} In addition, 18S RNA and 28S RNA were subjected to gel electrophoresis and confirmed by ETBR (ethidium bromide) staining. RNA concentrations were measured by A _260. Total RNA 2 μg 50U Super-Script Ⅱ reverse transcriptase (Invitrogen, Carlsbad, CA, USA), 5 μM DTT, 40U RNaseOUT recombinant ribonuclease inhibitor, 0.5 μmol of random hexanucleotide Reverse transcription was performed through 20 µl of a reaction solution containing a primer and a 500 µmol dNTP mixture. The reverse transcription reaction was performed at 50° C. for 60 minutes. Thereafter, the reaction solution was heated at 70° C. for 15 minutes to terminate the reaction, and the generated cDNA was stored at -20° C.

Example 8: quantitative real-time PCR ( quantitative real - time PCR ) analysis

In order to measure the effect of rhDPP on the differentiation of hDPC into oblasts, the mRNA levels of Col1, ALP, and DMP-1, which are mineralization markers for oblast differentiation, were measured. Expression of the gene was confirmed through quantitative real-time PCR using the cDNA obtained in Example 7 as a template. All real-time PCR was performed in the ABI Step One real-time PCR system. For each reaction, 0.1 μm of forward/reverse primer, 13.5 μl of 2-SYBR Green PCR master mix (Applied Biosystem, AmpliTaq Gold DNA polymerase and its buffer, dNTPs mixture, SYBR Green I dye, Rox dye, and 10 mm MgCl ₂ ) , And 15 µl of a reaction solution containing 1.5 µl template cDNA. In a typical amplification program, the enzyme was activated for 10 minutes at 94° C., followed by a step of denaturation at 94° C. for 15 seconds and annealing and elongation at 60° C. for 1 minute in one cycle and 40 times. . _{The C T} (cycle threshold) value for each gene was determined through the automatic limit analysis function of the ABI device _{, and the dC T} value was obtained by normalization by comparison with _{C T (GAPDH).} The difference in the number of n between the values of C _T or dC _T means that ^{there is a 2 n-fold difference between cDNA samples with the target sequence.} Primers used for quantitative real-time PCR are listed in Table 1 below.

Primers used for quantitative real-time PCR Gene Forward primer (5'-3') Reverse primer (5'-3') GAPDH TGGAAGGACTCATGACCACA
(SEQ ID NO: 4) TTCAGCTCAGGGATGACCTT
(SEQ ID NO: 5) Col1 GAAAATGGAGCTCCTGGTCA
(SEQ ID NO: 6) ACCCTTAGCACCAACAGCAC
(SEQ ID NO: 7) ALP CCAAAGGCTTCTTCTTGCTG
(SEQ ID NO: 8) CCACCAAATGTGAAGACGTG
(SEQ ID NO: 9) DMP-1 CCCAGTGACAGCACTCAGTC
(SEQ ID NO: 10) CTCCTTTTCCTGTGCTCCTG
(SEQ ID NO: 11)

As a result of checking the gene expression level of hDPC cultured with or without rhDPP treatment, the mRNA expression of DMP-1 did not show a significant difference when treated with rhDPP, but the mRNA expression of Col1 and ALP was compared to the untreated control group. Compared to the increase. In particular, the expression of ALP increased by about 1.6 times (Fig. 6). These results, together with the above results, suggest that rhDPP plays a role in the differentiation of hDPC oblasts.

Example 9: statistical analysis

Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed through the SAS program. Differences between the two groups were tested through the Student t-test. In all statistical analysis, the p value was less than 0.05, which was significant.

Taking the above results together, it is supported that rhDPP not only plays an important role in the differentiation of hDPC's oblastic cells, but also has a function of promoting proliferation and adhesion of pulp cells, so that it can be used for regeneration of teeth.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

<110> Industry-Academic Cooperation Foundation, Dankook University <110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Composition for adhesion or proliferation of dental pulp cells comprising recombinant dentin phosphoprotein <130> PA120995-KR-D1 <160> 11 <170> KopatentIn 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hDPP primer F <400> 1 tctcgaggat gatcccaata gc 22 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> hDPP primer R <400> 2 tggtaccatt agtctgatgt gctatc 26 <210> 3 <211> 58 <212> PRT <213> human DPP <400> 3 Asp Asp Pro Asn Ser Ser Asp Glu Ser Asn Gly Asn Asp Asp Ala Asn 1 5 10 15 Ser Glu Ser Asp Asn Asn Ser Ser Ser Arg Gly Asp Ala Ser Tyr Asn 20 25 30 Ser Asp Glu Ser Lys Asp Asn Gly Asn Gly Ser Asp Ser Lys Gly Ala 35 40 45 Glu Asp Asp Asp Ser Asp Ser Thr Ser Asp 50 55 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer F <400> 4 tggaaggact catgaccaca 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH primer R <400> 5 ttcagctcag ggatgacctt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Col1 primer F <400> 6 gaaaatggag ctcctggtca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Col1 primer R <400> 7 acccttagca ccaacagcac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALP primer F <400> 8 ccaaaggctt cttcttgctg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALP primer R <400> 9 ccaccaaatg tgaagacgtg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DMP-1 primer F <400> 10 cccagtgaca gcactcagtc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DMP-1 primer R <400> 11 ctccttttcc tgtgctcctg 20

Claims (7)

A composition for promoting adherence or proliferation of dental pulp cells comprising dentin phosphoprotein comprising the amino acid sequence of SEQ ID NO: 3 as an active ingredient.
The composition of claim 1, wherein the dentin phosphate protein is human.
delete A method for inducing attachment or proliferation of isolated dendritic cells, comprising treating dendritic phosphate protein consisting of the amino acid sequence of SEQ ID NO: 3 to isolated dendritic cells.
The dentin phosphate protein of claim 4, wherein the dentin phosphate protein is prepared by expressing recombinant human dentin phosphate protein in a host cell transfected with an expression vector comprising a base sequence encoding the amino acid sequence of SEQ ID NO: 3 Way.
6. The method according to claim 5, wherein the expression vector is a pBAD / His-A vector having the cleavage map shown in Fig.
5. The method of claim 4, wherein the dendritic cells are human dendritic cells.

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