KR101401718B1 - Composition comprising histone deacetylase inhibitor for dentin regeneration and its method - Google Patents

Abstract

The present invention discloses a pharmaceutical composition for promoting dentin regeneration comprising a histone deacetylase inhibitor selected from the group consisting of suberoylanilide hydroxamic acid, tricostatin A, verinostat and panominostat. The composition of the present invention can induce dentine cell differentiation and thus normal dentin formation, and thus can be useful for dentin regeneration-related diseases.

Description

[0001] The present invention relates to a composition for promoting dentin regeneration including histone deacetylase inhibitor,

The present invention relates to the field of treatment of dental reproduction-related diseases of teeth.

The tooth is an organs with an enamel on the outermost layer, a hard tissue called dentin in the inner layer, and dentin cells producing dentin on the inner side of the dentin, the center of which has the dimensions. Teeth can be lost due to caries or periodontal disease. The presence or absence of teeth greatly affects food consumption as well as appearance, and research on tooth restoration technology is underway to maintain health and maintain good quality of life.

The dentin is composed of dentine cells, which accounts for 80% of the teeth. Dentin provides a buffering effect when the enamel is energized and serves to transmit the stimulus to the dimension through the fine passage of the dentinal tubule. Although damage to dentin has partial regeneration capability, if the dentin is exposed by tooth decay or mechanical damage, there is a problem that the currently used treatment method can not make a regenerated dentin composed of normal dentin tissue at the exposed dimension . In other words, currently used dental prosthesis uses calcium hydroxide and the like to induce partial dentin necrosis and dentin formation in the lower part of the dentin, but the dentin is made into a tissue close to the bones, so that the function of the original dentin can not be sufficiently exhibited.

In addition, implants are mainly used to replace lost teeth. However, the purchase of the artificial root by the screw type titanium or the like used in the implant treatment has the disadvantage that the growth of the jaw bone in the jaw growth period is inhibited and the tooth can not move. To overcome this, research is underway to regenerate teeth.

Korean Laid-Open Patent Publication No. 2012-0058439 discloses a composition for regenerating teeth using a fusion protein comprising a fibronectin domain and an osteocalcin domain, and relates to a fusion protein for tooth and bone regeneration.

Korean Patent Laid-Open Publication No. 2004-0016449 relates to a material for regenerating a tooth, which comprises casein phosphoprotein (CPP) -amorphous calcium phosphate (ACP) complex or casein phosphopeptide (CPP) -amorphous calcium fluoride phosphate (ACFP) Disclosed is a composition for regenerating teeth comprising a complex.

Korean Patent Laid-Open Publication No. 2011-0045067 relates to a method for repairing a tooth defect portion and a method for manufacturing a restoration material, and discloses a composition for dental restoration using cells comprising at least one of mesenchymal stem cells derived from a tooth root and epithelial cells do.

The present invention provides a method for promoting the regeneration of dentin tissues in a tooth region by inducing dentin cross-linking so that the function of the original dentin is fully exerted or the replacement of lost teeth is promoted.

In one embodiment, the present invention provides a pharmaceutical composition for promoting dentin regeneration comprising a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is selected from the group consisting of suberoylanilide hydroxamic acid (hereinafter referred to as 'SAHA'), A, verinostat and panominostat.

 The present invention also provides a pharmaceutical composition for promoting dentin regeneration, wherein the dentin regeneration according to the present invention is by promoting the differentiation of dermal stem cells into dentine cells.

In another aspect, the present invention provides a method of producing diaoblast cells comprising treating a stem cell with a histone deacetylase inhibitor according to the present invention, wherein the histone deacetylase inhibitor is selected from the group consisting of SAHA, tricostatin A, Nostart, and panominostat.

The present invention also provides a method of generating an osteoblast, the method further comprising the step of detecting the differentiation markers into the dendritic cells.

The present invention also provides a method of producing an oocyte, wherein the markers of differentiation into dendritic cells according to the present invention are nestin, alp, Dmp1, or Dspp, wherein the expression of the gene is increased in the i.v.

In another aspect, the present application provides an ivocobblast produced by the method of producing an oocyte according to the present invention.

In another aspect, the present invention provides a dentin regeneration-promoting composition comprising an osteoblast according to the present invention.

In yet another embodiment, the present invention provides an ivocyte that is used in pulpotomy or pulpectomy procedures, or is used adjunctively to a pulpotomy procedure.

The composition comprising the histone deacetylase inhibitor of the present invention was found to promote the differentiation of dendritic cells in dendritic cells and promote the formation of calcified nodules to promote dentin formation. Based on these results, the compositions and methods of the present invention can produce regenerated dentin closer to dentin tissue than bone tissue and can be effectively used for tooth restoration or regeneration.

FIG. 1A shows the cytotoxicity of SAHA as a histone deacetylase inhibitor. FIG. 1B and FIG. 1D show the results of observing the formation of calcified crystals after treatment of MDPC23 and OD11 cell lines capable of differentiating SAHA into dAb Fig. 1C shows the results of observing whether truncated nodule formation was promoted after treating MDC23 cell line with tricostatin A. Fig.
FIG. 2 shows the results of quantitative PCR for the expression of the differentiation marker gene of the iooblast when the SAHA treatment was performed. As a result, mRNA expression of the differentiation marker genes nestin, alp, Dmp1 and Dspp is promoted.
FIG. 3 shows the expression of Nfic, a transcription factor that promotes transcription of Dspp, which is one of the differentiation markers of dendritic cells, and shows that Nfic mRNA and protein expression are increased by SAHA.
FIG. 4 shows that SAHA increased NFic binding to the Dspp promoter and promoted Dspp transcription (FIG. 4 a), demonstrating that SAHA increases Dspp promoter binding alone or with overexpressed Nfic 4 b).
FIG. 5 shows the results of observing the disappearance of expression promoting activity of sarcoma differentiation markers by SAHA in the transcription (FIG. 5A) and the protein level (FIG. 5B) in the decrease of Nfic expression.

The present invention relates to a novel use of a histone deacetylase inhibitor. It has been confirmed that a composition comprising a histone deacetylase inhibitor promotes dAb regeneration by inducing differentiation of dermal cells into dermal cells, thereby completing the present invention .

In one aspect, the present invention relates to a pharmaceutical composition for promoting dentin regeneration comprising a histone deacetylase inhibitor.

As used herein, the term " tooth " is a rigid calcified structure located within the oral cavity and is used to chew food. Its structure includes an enamel layer on the outer side and dentin on the inner side of hard tissue, to be.

The term " dentin " as used herein refers to a tissue in which dentin cells producing dentin are present in the inside of the tooth, accounting for 80% of the teeth. The dentin can be identified by the presence of dentin cells, and the presence of dentin cells can be confirmed by the presence or absence of dentin cyerioprotein. Identification of dendin cyhaloproteins can be easily carried out by well-known methods in the art, for example, in situ hybridization, antibody staining, and the like. The dentin is a porous tissue that contains many dentine tubules with a hard calcified tissue consisting of about 70% hydroxyapatite, 20% collagen, and 10% water. It is placed between the enamel and the scales to protect the dimensions, It has a major mechanical function in that.

As used herein, the term " diaoblast " is a kind of mesenchymal stem cells that are differentiated from mesenchymal mesenchymal stem cells derived from neural ridocytes during development, and thereafter are cells that can be differentiated from mesenchymal stem cells to be. The dentin cells form a layer of cells at the interface between the dentin and the dentin and produce dentin.

The histone deacetylase inhibitor is a compound that inhibits the function of histone deacetylase and has been used as a mood stabilizer and an antiepileptic agent in psychiatric and neurological disorders and a known conventional histone deacetylase inhibitor can be used herein have.

The histone deacetylase inhibitors that can be used herein include not only traditional inhibitors such as hydroxamic acids, prototypic peptides, benzamides, protonic ketones and aliphatic acids, but also hydroxides as second generation histone deacetylase inhibitors And more specifically, suberoylanilide hydroxamic acid, or vorinostat or N-hydroxy < RTI ID = 0.0 >-N'-phenyl-octanediamide, SAHA), trichostatin A, 7- [4- (dimethylamino) phenyl] -N-hydroxy-4,6-dimethyl-7-oxohepta-2,4- 2-enamide} and Panominostat {Panobinostat, (2E) -N-hydroxy-3- [4 (4-fluorophenyl) - ({[2- (2-methyl-1H-indol-3- yl) ethyl] amino} methyl) phenyl] acrylamide} is.

The histone deacetylase inhibitor of the present invention may be used as such or in various pharmaceutically acceptable salt forms. As used herein, " pharmaceutically acceptable salts " refers to those which are physiologically acceptable and which, when administered to humans, do not normally cause an allergic reaction such as gastrointestinal disorders, dizziness, or the like, By reacting a stoichiometric amount of an appropriate base or acid in a solvent or a mixture of these two solvents. These include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids and salts with basic or acidic amino acids. Salts with inorganic bases include, for example, alkali metal salts such as sodium salts and potassium salts, alkaline nasal metal salts such as calcium salts and magnesium salts, aluminum salts and ammonium salts. Examples of salts with organic bases include salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N, And salts with dibenzylethylenediamine. Examples of salts with inorganic acids include salts with hydrochloric acid, boric acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methylsulfonic acid, benzylsulfonic acid or p- toluenesulfonic acid. Salts with basic amino acids include, for example, arginine, lysine and ornithine. Examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid. Salts according to the invention can be prepared by conventional methods such as, for example, ion exchange.

The dentin bones regenerated by conventional dental implantation have features similar to bone in mineral content and the like, and are in a structure close to the bones rather than dentin. Thus, the dentin bone can not sufficiently exhibit the functions of the original dentin. (Goldberg M et al., Dentin extracellular matrix molecules implanted into exposed pulps to generate reparative dentin: a novel strategy in regenerative dentistry. J Dent Res 88: 396-9, 2009)

Diseases that can be prevented or treated with the composition of the present application include diseases requiring dentin regeneration, for example, regeneration of missing teeth as well as tooth decay, dental congestion, dimensional alteration, dimensional necrosis, apical lesion or root apex Including, but not limited to,

The compositions herein can also be used as adjuncts as a component of a repellent for use in pulpotomy or pulpectomy procedures or in dental pulp surgery.

 When the composition of the present disclosure is prepared with a pharmaceutical composition, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be included in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid But are not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil no. The pharmaceutical composition of the present invention may further comprise a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, and a preservative in addition to the above components.

Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed. 1995).

The pharmaceutical composition of the present invention can be administered parenterally, and in the case of parenteral administration, it can be applied by local administration, that is, by direct application or injection, particularly to a dental site requiring dentin regeneration, but other administration methods are not excluded.

Suitable dosages of the pharmaceutical compositions herein will vary depending on factors such as the particular type of disease and method of administration, the mode of administration, the age, weight, sex, pathology, food, time of administration, route of administration, rate of excretion, And the like.

The concentration of the histone deacetylase inhibitor in the composition according to the present invention may be varied depending on the kind of the inhibitor and may be about 1 nM to 20 μM, preferably about 1 nM to 10 μM, more preferably about 1 nM to 5 μM As shown in FIG.

The composition of the present invention may further contain one or more additional active ingredients exhibiting the same or similar functions.

The compositions herein may further comprise one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. Examples of pharmaceutically acceptable carriers include saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, combinations thereof and the like, and if necessary, an antioxidant, a buffer, And other conventional additives may be added. In addition, a diluent, a dispersant, a surfactant, a binder, and a lubricant may be additionally added to formulate the composition for injectable use such as an aqueous solution, a suspension or an emulsion. Further, it can be suitably formulated according to each disease or component according to a suitable method in the art or by a method disclosed in Remington's Pharmaceutical Science (recent edition, Mark Publishing Company).

The composition of the present invention can be used for dentin regeneration alone or in combination with a method of using surgery, drug treatment and biological response modifiers.

In another aspect, the present invention relates to a method of producing dental < RTI ID = 0.0 > embryo, < / RTI > comprising the step of treating a histone stem cell with a histone deacetylase inhibitor.

The method comprises treating a stem cell with a histone deacetylase inhibitor according to the present invention; Culturing the treated stem cells in an epiblast differentiation medium for about 2 to 20 days, particularly about 7 to 14 days, in an in vitro or in vitro manner. When performed in Invitro, the dental bone cells can be differentiated from the mesenchymal stem cells or bone marrow mesenchymal stem cells obtained from the subject and then re-implanted in the same subject.

As used herein, the term "stem cell" refers to a cell having the ability to divide mesenchymal cells into dentine cells by external stimulation to form dentin during undifferentiation in the dimension. Dimensions Stem cells can be obtained by separating the dimensions from the picked or permanent teeth and then culturing them.

The epithelial differentiation medium can promote the differentiation of dendritic cells into dendritic cells, for example, those described in the Examples herein can be used.

The method may further comprise the step of identifying the dentine cells formed by the method. Identification of the dAb cells may be carried out through the detection of differentiation marker markers, for example, nestin, alp (alkaline phosphatase), Dmp1 (dentin matrix protein 1) and / or Dspp (dentin sialophorase For example, by detecting the presence or absence of the Nfic gene, or by detecting the expression of the gene, for example, by detecting the Nfic gene expression.

Expression of a gene includes both expression at the transcription and translation levels. For example, after reverse transcription, PCR, quantitative PCR or real-time quantitative PCR, or Northern blotting or Western blotting, or Western blotting or Western blotting, may be carried out by known methods in the art. Or a microarray method (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)).

In another aspect, the present invention also relates to a dentin regeneration-promoting composition comprising the above-described dentin cells and the above-described dentin-derived cells.

The cellular composition may be administered parenterally, and various biocompatible materials such as polysaccharides, monosaccharides, polymeric organic materials, and minerals may be used in addition to or in place of materials that may be included in the pharmaceutical compositions described above.

Hereinafter, the present invention will be described in more detail by way of examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the subject matter of the present invention.

Example  One : SAHA of In the oocyte  Of Calcified Nodule Formation

1-1 Measurement of cell viability cell viability assay )

In order to determine the cytotoxicity of SAHA, MDCPC23 cells (Hanks et al., Cloned 3T6 cell line from CD-1) derived from mouse dental papillae at a concentration of 2 x 10 ³ per well in 96- (Dulbecco's modified Eagle's medium (DMEM; Hyclon, USA) containing 10% fetal bovine serum) and 24 h after SAHA (1, 3, 5 mu M) was added. After 48 h of incubation, cell viability was investigated using Ez-CyTox (Daeil Lab Service, Korea) according to the manufacturer's instructions. As shown in Fig. 1A, it was confirmed that there was no toxicity to the cells.

1-2 Calcified nodule formation

The cells were cultured in a culture medium containing 10% fetal bovine serum, 10 mM beta-glycerophosphate, 50 μg / ml bovine serum albumin, ml ascorbic acid). MDPC23 cells were cultured in the presence of SAHA and tricostatin for 14 days and in the presence of SA11 for 14 days at 37 ° C. for 7 days. Alizalin red S, Sigma-Aldrich, USA ) Staining. The calcification nodule identification test using Alizarin Red S was carried out by the method of the manufacturer, in which the production of calcified dentin, which is the final product of the differentiation of the dental epithelium, was confirmed by Invitro.

In order to quantify the degree of calcification, alizarin red S deposited on a calcified substrate was dissolved in acid (HCl (hydrogen chloride) containing 5% SDS (sodium deoxycholate)) and absorbance was measured at 405 nm See chart).

As a result, it can be seen that SAHA increases the formation of calcified nodules in MDPC23 cells (Fig. 1b) and OD11 cells (Fig. 1d) in a dose dependent manner, as shown in Figs. 1b and 1d. As a result, it was also found that trichostatin A (FIG. 1c) also increased the formation of calcified nodules in a dose-dependent manner in MDPC23 cells.

Example  2 : SAHA On by Epithelial cells  Promote differentiation marker gene expression

The MDPC23 cell line was cultured for 7 days in an epicatechin differentiation medium containing SAHA at the concentration shown in FIG. 2, and then the expression of nestin, alp, Dmp1 and Dspp was detected by reverse transcription PCR. Total RNA was extracted from the cells using an easy-BLUE ^™ RNA extraction reagent (iNtRON Biotechnology, Korea) according to the manufacturer's method, and the extracted RNA was purified using AccuPower ^™ RT-PreMix (Bioneer, Korea) After cDNA synthesis, PCR was carried out using the following primers (denaturation at 95 ° C, annealing at 58 ° C, elongation at 72 ° C: alp, dmp 1 33 cycles, nestin 40 cycles). The primer sequences are: nestin-forward (f) 5'-CCCTGAAGTCGAGGAGCTG-3 ', nestin-reverse (r) 5'-CTGCTGCACCTCTAAGCGA-3'; alp-f 5'-AGGCAGGATTGACCACGG-3 ', alp-r 5'-TGTAGTTCTGCTCATGGA-3', Dmp1-f 5'-AACTGGAAGTGATGAGGAC-3 ', Dmp1-r 5'-TTTAGATTCTTCCGACCTGA-3'; Gapdh-f 5'-TCACCATCTTCCAGGAGCG-3 'Gapdh-r 5'-CTGCTTACCACCTTCTTGA-3'. The primer sequences for quantitative Real Time-PCR are: Nfic-f 5'-GACATGTACCTGGCCTACTTTG-3 ', Nfic-r 5'-CACACCTGACGTGACAAAGCTC-3'; Dspp-f 5'-ATTCCGGTTCCCCAGTTAGTA-3 ', Dspp-r 5'-CTGTTGCTAGTGGTGCTGTT-3'; And Gapdh-f 5'-TCAATGACAACTTTGTCAAGC-3 'Gapdh-r 5'-CCAGGGTTTCTTACTCCTTGG-3'. The difference between the samples was corrected by the amount of GAPDH expression. Data are presented as relative transcript concentration.

The results are shown in FIG. The left photograph shows the results of RT-PCR analysis of nestin, alp and Dmp1 with agarose gel, and the right graph shows the quantitative PCR results of Dspp transcript as a relative ratio to Gapdh. As shown in Fig. 2, mRNA expression of nestin, alp, Dmp1, and Dspp was increased as compared with the case of not treating SAHA, indicating that it was differentiated into dAb cells by SAHA treatment.

Example  3: SAHA of Epithelial cells  Promoting differentiation Nfic Correlation with

3-1 SAHA of Nfic  Analysis of Expression Promotion Effect

The transcriptional and translational levels of Nfic, which is known as a transcription factor promoting the transcription of Dspp, a representative differentiation marker gene, were confirmed by quantitative real time PCR and Western blot analysis, respectively.

The Nfic quantitative PCR assay method and primers are as described in Example 2. In the case of Western blotting, the MDPC23 cell line was treated with 5 μM of SAHA for 48 hours. Then, the cells were treated with 50 mM Tris [pH 7.5], 150 mM NaCl, 1% Tween 20, 0.2% NP-40, 1 mM phenylmethylsulfonyl fluoride, Cells were thawed on ice with a lysis solution consisting of 100 mM NaF, 1 mM Na ₃ VO ₄ and 1 tablet / 10 ml protease inhibitor cocktail tablet (Roche). Proteins were heated for 5 minutes in 5 X SDS sample buffer, proteins were separated by electrophoresis on SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride (PVDF) cell membranes (Millipore). After this transfer, the cell membrane was blocked with skim milk and anti-NFI-c, a primary antibody polyclonal antibody (Lee D et al., Nuclear factor IC is essential for odontogenic cell proliferation and (Sigma-Aldrich) and Supex reagent (Sigma-Aldrich) were incubated with the horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) (Dyne-Bio) and visualized with LAS1000 (Fuji PhotoFilm). In Western blot, the correction of the inter-sample differences was corrected by the amount of beta actin expression.

The results are shown in FIG. The left graph shows the Western blotting results and the right graph shows the relative expression of the Nfic transcripts. As shown in the figure, mRNA and protein expression of Nfic, a transcription factor that promotes transcription of Dspp, a marker gene for differentiation of dAb, is increased by SAHA.

3-2 Nfic of Dspp  Promoter Binding Assay

In addition, a luciferase reporter activity assay and a promoter binding assay, including the Dspp promoter, were performed to determine whether Nfic increased by SAHA actually binds to the Dspp promoter and promotes Dspp transcription.

For the luciferase reporter activity assay, MDPC23 cells were seeded into 96-well plates at 1 × 10 ⁴ cells per well and cultured overnight. After transferring the following plasmids using lipofectamine (Invitrogen, USA), SAHA (5 μM) . ≪ / RTI > The plasmid used was as follows: 0.2 μg of Nfic expression vector (pCH-Nfic: Lee D et al., Nuclear factor IC is essential for odontogenic cell proliferation and odontoblast differentiation during tooth root development. J Biol Chem 284: 17293-303, 2009), 0.2 μg of reporter vector (pGL3-basic (without promoter), pGL3-Dspp-luc, or pGL3-Dspp-MT-luc (pGL3-Dspp-luc) containing the Dspp promoter PGL3-Dspp-MT-luc was obtained from pGL3-Dspp-luc through a site-specific mutation that changed the base sequence to CTGAT from -1021 to -1017 bp corresponding to -1021 to -1017 bp of the Dspp promoter site. Cosmogenetech, Cells were harvested 24 h later and analyzed using the Dual-Glo luciferase assay kit (Promega, USA) according to the manufacturer's instructions. As a control group, The base of the NFic binding site A Dspp promoter containing the mutation (MT) in the sequence was used.

The results are shown in Figure 4a. As shown in the left graph of FIG. 4A, it can be seen that the activity of Dspp promoter reporter in the presence of SAHA or NFic overexpression was markedly enhanced as compared with the control group without any treatment. In contrast, the modulatory effect of SAHA using the Dspp-MT-luc mutant with the Nfic binding site of the promoter was observed (right graph in FIG. 4A), indicating no increase in luciferase activity.

Nfic binding to the Dspp promoter was then carried out using chromatin immunoprecipitation (ChIP). pcDNA expressing pcDNA or Nfic was transfected into MDPC23 cells as described above and then cultured for 48 hours in the presence or absence of SAHA. After the cells were treated with 1% formaldehyde and melted, sonication was performed. Protein G agarose beads were previously pretreated with Nfic antibody or immunoprecipitated with rabbit IgG as a control. The DNA was then isolated and PCR was performed (denaturation at 95 ° C, annealing 54) using primers (f 5'-GCAAAGTGTTTACTATGGAAG-3 'and r 5'-CACTATCCAGCAACAGCATC-3') at positions corresponding to -1083 to -947 bp of the Dspp promoter ℃, elongation 72 ℃; 40 cycles) and then electrophoresed on agarose gel.

The results are shown in Figure 4b. In the presence of SAHA, the binding of the Nfic protein to the Dspp promoter is markedly increased. SAHA increased Dspp promoter binding alone or with overexpressed Nfic (Figure 4b). These results suggest that the expression of Nfic is involved in the promotion of Dspp expression by SAHA and thus the transcription factor activity of Nfic is involved.

3-3 RNA  By interference Nfic Lt; / RTI > SAHA of Epithelial cells Differentiation marker  Effect on gene expression

When the expression of Nfic was reduced using siRNA in MDPC23 cells, the above-mentioned SAHA stimulated the expression of the differentiated markers of dAb, and this was examined by the following method.

ON-TARGET plus SMART full mouse Nfic siRNA (L-062258-00-0005) and ON-TARGET plus non-targeting siRNA (control siRNA, D-001810-02-05) were purchased from Dharmacon (Lafayette, CO, USA) Respectively. The siRNA was transferred to MDPC23 cells using the method of Dharmafect (Dharmacon), and after 48 hours, the interference efficiency was evaluated by real-time PCR and Western blot analysis. Non-target siRNA was used as a control.

Quantitative PCR and Western blotting were performed as described in Examples 2 and 3-1.

The results are shown in FIG. As shown in FIG. 5 (a), inhibition of the expression of Nfic by siRNA indicates that the effect of increasing the expression of Dspp, Dmp1, and nestin in SAHA disappeared at transcription (FIG. 5a) and at the protein level (FIG. 5b). These results suggest that the stimulation of SAH - induced differentiation of the epithelial cells is Nfic - dependent.

Claims (10)

Wherein the suboyl anilide hydroxamic acid increases the expression of Nfic (Nuclear Factor IC) protein in the cell, and the Nfic promotes the transcription of Dspp (Dentin sialophosphoprotein) Wherein the pharmacologically acceptable salt thereof is a pharmacologically acceptable salt thereof.
delete The pharmaceutical composition for promoting dentin regeneration according to claim 1, wherein the cell is a mesenchymal stem cell or bone marrow mesenchymal stem cell, and the composition is promoted by differentiation of the cell into diaoblast.
Comprising the step of treating subcutaneous stem cells or mesenchymal mesenchymal stem cells in the Invitro with suberoylanilide hydroxamic acid.
5. The method of claim 4,
Wherein the suboyl anilide hydroxamic acid increases the expression of Nfic (Nuclear Factor IC) protein in the mesenchymal stem cells of the stem cell or the bone marrow, and the Nfic promotes the transcription of Dspp (Dentin sialophosphoprotein) Differentiation of dentate germ cells.
5. The method of claim 4, wherein the method further comprises detecting a differentiation marker gene into an epithelial cell.
[Claim 7] The method according to claim 6, wherein the differentiation marker gene of the dentate gland is nestin, alp, Dmp1, or Dspp, and the expression of the gene is increased in the i.v.
delete delete 5. The method of claim 4, wherein the method is used in pulpotomy or pulpectomy procedures, or is used adjunctively in the pulpotomy procedure.

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