Polypeptide sequence designed based on AP2α sequence and functional regulation use for odontogenic mesenchymal stem cells

Abstract

For a specific peptide sequence through which AP2α protein binds to a BARX1 promoter region, a small-molecule peptide is researched, developed and utilized by simulating a binding site sequence, so that a binding site for AP2α on a BARX1 promoter can be specifically blocked, the binding of an AP2α complex to the BARX1 promoter can be blocked, an inhibitory effect of AP2α on BARX1 transcription can be alleviated, and BARX1 transcription can be promoted, thereby finally realizing the effects of promoting odontogenic differentiation of odontogenic mesenchymal stem cells and dentin regeneration mediated by odontogenic mesenchymal stem cells within the jawbone, thus improving the success rate of biological tooth root regeneration.

Description

Polypeptide sequence designed based on AP2 alpha sequence and application of regulating function of dental mesenchymal stem cells

[0001] The present application claims priority to the Chinese patent application No. 2024111545392, filed on August 21, 2024, and entitled "Polypeptide sequence designed based on AP2 alpha sequence and application of regulating function of dental mesenchymal stem cells", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biomedicine, in particular to polypeptide based on AP2 alpha sequence and application of regulating function of dental mesenchymal stem cells. BACKGROUND

[0003] Tooth loss is a common and frequently-occurring disease in the oral cavity, with a morbidity rate ranking first among human organ diseases, which seriously affects the physiological functions of patients such as mastication, swallowing, and pronunciation, and also increases the mortality rate of upper gastrointestinal cancer, heart disease, and stroke. The existing repair methods are all non-physiological denture repair, which cannot completely restore the structure and function of normal teeth. For example, the current implant denture is the best repair method for tooth loss recognized by the clinic, but due to the lack of physiological structures such as periodontal membrane, its occlusal force perception and occlusal force dispersion ability are poor, in addition, there are complications such as peri-implantitis. With the development of stem cell and tissue engineering technology, the use of biotechnology to achieve functional tooth regeneration will become a new direction for the future treatment of tooth loss.

[0004] Current tooth regeneration includes two ways, one is whole tooth regeneration based on developmental principles, but there are some problems in current whole tooth regeneration. First, it is difficult to obtain seed cells, and it is necessary to find cells with tooth induction effect and capable of reproducing epithelial-mesenchymal interaction in whole tooth regeneration process, which is the basis for developing whole tooth regeneration. Second, it is necessary to find programmed key genes to make epithelium and mesenchyme differentiate in their respective directions. In addition, problems such as tooth shape, growth and eruption control, and immune rejection of allogeneic transplantation hinder the development of whole tooth regeneration, so it is not clear when whole tooth regeneration can be applied. The other is tooth regeneration based on stem cell and tissue engineering technology. Compared with whole tooth regeneration, tissue engineering tooth regeneration temporarily avoids the technical difficulties of tooth shape control and eruption control, and can be more quickly applied in clinical transformation. Tooth root is the basis for tooth function, and some scholars have successfully applied apical tooth papilla stem cells, dental pulp stem cells and periodontal ligament stem cells to achieve biological tooth root regeneration in small pig animal models. Although some progress has been made in the study of biological tooth root, there are still some problems that need to be further studied and solved, the most critical of which is the low success rate, which limits its clinical transformation application. How to improve the success rate of biological tooth root regeneration is a problem that needs to be solved to promote its clinical transformation.

[0005] To achieve this goal, scholars from all aspects of tissue engineering technology research. Because of the kinship of tissue origin, dental mesenchymal stem cells have stronger tooth differentiation ability than non-dental mesenchymal stem cells, which is undoubtedly the best choice for seed cells. Literature reports that apical dental papilla stem cells can not only stimulate the formation of tooth pulp-like tissue in the root canal and the deposition of dentin-like tissue on the root canal wall, but also can induce the regeneration of dentin-like tissue in large animal experimental models. In the scaffold aspect, some scholars use dentin matrix as a scaffold material to simulate the microenvironment of dentin regeneration, and some scholars use a sandwich composite of PLGA / gelatin electrostatic membrane, dentin matrix and dental pulp extracellular matrix arranged in turn as a scaffold, which can form a good microenvironment for tooth root regeneration. However, it is also reported that dentin matrix has good bone conduction ability and can be used for bone tissue regeneration. In addition, studies have found that many genes have the function of promoting the tooth differentiation of stem cells, such as BMP2, 4, 7, KDM6B, SMAD4, SHANK2, BCL2, SFRP2, TET1, FHL2, PAX9, OSX, DMPs and DSPP. However, these genes either promote osteogenic and odontogenic differentiation functions at the same time, or promote odontogenic differentiation but the effect on osteogenic differentiation is unclear, and lack evidence of promoting odontogenic and inhibiting osteogenic differentiation. Because biological tooth root regeneration occurs in the jawbone microenvironment, the jawbone microenvironment tends to induce stem cell bone differentiation rather than tooth differentiation. Therefore, if the key gene with the function of promoting odontogenic differentiation and inhibiting osteogenic differentiation can be found, it will effectively promote the odontogenic differentiation function of stem cells in the jawbone microenvironment and improve the success rate of biological tooth root. Through the comprehensive analysis and research of normal and abnormal development of teeth combined with the differentiation potential of dental mesenchymal stem cells, the key target and molecular mechanism for promoting the regeneration of tooth roots in the jawbone will be found, the odontogenic and osteogenic differentiation processes can be accurately regulated, and thus a theoretical basis and candidate target for improving the efficiency of biological tooth root regeneration is provided. On this basis, small molecule preparations or drugs for regulating odontogenic differentiation are developed to promote their clinical translation and application. SUMMARY

[0006] Therefore, the present application provides a polypeptide based on the sequence of AP2α and its application in regulating the function of dental mesenchymal stem cells.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a polypeptide having:

[0009] (I) an amino acid sequence as shown in any one of SEQ ID No. 3-13;

[0010] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence according to (I) and having the same function as the amino acid sequence according to (I); or

[0011] (III) an amino acid sequence having 90% or more identity with the amino acid sequence according to (I) or (II).

[0012] In a second aspect, the present application further provides use of the polypeptide in preparation of a bioactive peptide for interfering the binding of AP2α protein to BARX1 promoter.

[0013] In a third aspect, the present application further provides a bioactive peptide having:

[0014] (I) an amino acid sequence as shown in SEQ ID No. 1 or 2;

[0015] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence according to (I) and having the same function as the amino acid sequence according to (I); or

[0016] (III) an amino acid sequence having 90% or more identity with the amino acid sequence according to (I) or (II).

[0017] In a fourth aspect, the present application further provides use of the bioactive peptide in preparation of a reagent or a drug for any one of:

[0018] (I) specifically blocking the binding site of AP2α on BARX1 promoter;

[0019] (II) blocking the binding of AP2α complex to BARX1 promoter;

[0020] (III) reducing the transcriptional inhibition of AP2α on BARX1;

[0021] (IV) promoting the transcription of BARX1.

[0022] In a fifth aspect, the present application further provides use of the bioactive peptide in preparation of a reagent or a drug for promoting the increase of BARX1 / OSX protein complex binding in odontogenic mesenchymal stem cells.

[0023] In a sixth aspect, the present application further provides use of the bioactive peptide in preparation of a reagent or a drug for promoting the odontogenic differentiation of odontogenic mesenchymal stem cells.

[0024] In a seventh aspect, the present application further provides use of the bioactive peptide in preparation of a reagent or a drug for mediating dentin regeneration and / or biological root regeneration of dental pulp stem cells in the jaw.

[0025] In an eighth aspect, the present application further provides use of the bioactive peptide in preparation of a reagent or a drug for improving success rate and / or regeneration effect of biological root regeneration.

[0026] In some embodiments of the present application, the dental mesenchymal stem cells comprise apical papilla stem cells or dental pulp stem cells.

[0027] In a ninth aspect, the present application further provides a reagent or a drug comprising the bioactive peptide.

[0028] The present application is based on previous studies and clinical application, and clarifies how to regulate the role of BARX1 to promote odontogenic mesenchymal stem cell differentiation under specific clinical conditions, inhibit stem cell osteogenic differentiation, and promote odontogenic mesenchymal stem cell-mediated dentin regeneration in the jaw, thereby improving the success rate of biological root regeneration. This study will help to clarify the molecular mechanism of odontogenic mesenchymal stem cell directional differentiation and functional regulation. Through the development of new small molecule preparations, this study will provide a theoretical basis and key target for the functional reconstruction of odontogenic mesenchymal stem cells and the improvement of biological root success rate, and provide a basis for its clinical application. Findings:

[0029] Based on the mechanism research, the specific peptide segment sequence of AP2α protein binding to the BARX1 promoter region is targeted, and the binding site sequence is simulated to develop and use small molecule polypeptides. The binding site of AP2α protein on the BARX1 promoter can be specifically blocked, the AP2α complex is combined to the BARX1 promoter, the transcriptional inhibition of AP2α on BARX1 is reduced, the transcription of BARX1 is promoted, and finally the effects of promoting odontogenic mesenchymal stem cell differentiation, inhibiting osteogenic differentiation, and promoting odontogenic mesenchymal stem cell-mediated dentin regeneration in the jaw are achieved, thereby improving the success rate of biological root regeneration.

[0030] including but not limited to:

[0031] 1. The bioactive polypeptides AP2α-26 and AP2α-27 inhibit odontogenic mesenchymal stem cell osteogenic differentiation and promote apical papilla stem cell odontogenic differentiation.

[0032] 2. The bioactive polypeptides AP2α-26 and AP2α-27 promote the effect of odontogenic mesenchymal stem cell-mediated dentin regeneration in the jaw of rabbits, thereby improving the success rate of biological root regeneration. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments or prior art description will be briefly introduced below.

[0034] Figure 1 shows that the polypeptide microarray technology is used to find the DNA binding site of AP2α protein and BARX1; wherein, a: shows the results of immunological hybridization experiment, the upper graph is the result of nucleic acid hybridization, and the lower graph is the negative control without nucleic acid probe; the polypeptide microarray chip is made of BARX1 DNA and incubated with AP2α, and 11 positive polypeptide binding sites are found; b: shows the polypeptide microarray chip and nucleic acid probe, and the data analysis graph of detection color spot (the positive film point optical density value of AP2α corresponding array;); wherein, the abscissa is the polypeptide point number, that is, the 45 polypeptides corresponding to the array, and the ordinate is the percentage of point optical density value; c-d: the results of Western blot detection and gray value analysis show that only AP2α-26 and AP2α-27 in AP2α-26, AP2α-27 and AP2α-36 can open the binding of AP2α protein to the DNA promoter region of BARX1, block the inhibitory effect of AP2 protein on BARX1, and make the expression of BARX1 increase; **P≤0.01;

[0035] Figure 2 shows that the bioactive polypeptides AP2α-26 and AP2α-27 increase the binding of BARX1 / OSX protein complex in apical herticle stem cells; wherein, the results of western blot show that after adding AP2α-26 and AP2α-27 in apical herticle stem cells, the expression of BARX1 and OSX increases, and the results of Co-IP show that AP2α-26 and AP2α-27 increase the binding of BARX1 / OSX protein complex in apical herticle stem cells;

[0036] Figure 3 shows that the bioactive polypeptides AP2α-26 and AP2α-27 increase the binding of BARX1 / OSX protein complex in New Zealand rabbit dental pulp stem cells; wherein, the results of western blot show that after adding AP2α-26 and AP2α-27 in New Zealand rabbit dental pulp stem cells, the expression of BARX1 and OSX increases, and the expression of osteogenic marker gene BSP decreases; the results of Co-IP show that AP2α-26 and AP2α-27 increase the binding of BARX1 / OSX protein complex in New Zealand rabbit dental pulp stem cells;

[0037] Figure 4 shows that the number of new tooth-like tissues in the extraction socket of New Zealand rabbits is increased after the extraction socket is implanted with the bioactive polypeptide AP2a-26 and AP2a-27 pre-treated human apical hTSCs; wherein, a-b: the general view, micro-CT and quantitative analysis show that there are new tooth-like tissues in the extraction socket of the AP2a-26 group, there are new tooth-like tissues in the extraction socket of 4 rabbits of the AP2a-27 group, and there is new tooth-like tissue in the extraction socket of 1 rabbit of the Control group, and there is no obvious new tooth-like tissue in the extraction socket of the Mock group and the Matrigel group; c-d: the scanning electron microscope and HE staining results show that a large number of dentin tubules arranged regularly can be seen in the AP2a-26 group and the AP2a-27 group, and the dentin tubules arranged in the Control group are less regular; e-f: the immunohistochemical and quantitative results show that the expression of DSPP in the AP2a-26 group and the AP2a-27 group is increased, *P≤0.05, **P≤0.01;

[0038] Figure 5 shows the schematic diagram of the polypeptide chip on the polypeptide array film and the staining diagram after synthesis; wherein, the upper diagram is a schematic diagram; the lower diagram is an array staining diagram, and the staining depth does not represent the polypeptide concentration;

[0039] Figure 6 shows the sequences with obvious differences;

[0040] Figure 7 shows the polypeptide sequences of the polypeptide chip. DETAILED DESCRIPTION

[0041] The polypeptide sequence of AP2a and the application of regulating the function of odontogenic mesenchymal stem cells are disclosed, and those skilled in the art can refer to the content of the present application and appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the related personnel can obviously modify or appropriately change and combine the method and application described in the present application without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0042] The research of regeneration of any tissue or organ must be based on the deep understanding of its development mechanism. The signal molecules regulating tooth development are extremely complex. In addition to the regulation of normal tooth development, the abnormal tooth development regulation mechanism can also provide reference for tooth regeneration. Therefore, the research of tooth development principle and the regulation of tooth differentiation potential of dental stem cells can help us to find the key molecules promoting tooth in the jaw. BARX1 is a member of the BARX family of genes, which is a transcription factor expressing homologous domains. Studies have shown that BARX1 is involved in the regulation of tooth morphogenesis and neural crest-related maxillofacial development, and the knockout of BARX1 gene stops tooth development. In addition, studies have shown that Hoxa2 can inhibit the expression of BARX1 during the development of the palate of mice, and BMP4 shows inhibition of BARX1 during molar development, and the non-canonical Wnt family member Wnt5a inhibits the transcription of BARX1 during human tooth development, FGF8 plays an important role in maintaining the expression of BARX1 during the development of zebrafish gill arch, and BARX1 is a direct downstream gene of GATA4 in mouse neural crest cells, which is positively regulated by GATA4. The above studies show that BARX1 is an important transcription factor in tooth development. In addition, studies have found that miR-7a and miR-203 inhibit the expression of BARX1 in mouse gastric mesenchymal cells, but the functional regulation of BARX1 in mesenchymal cells is not clear. Our group found that the expression of BARX1 was abnormally increased in the gene expression profile of apical tooth papilla stem cells of patients with excessive root development in the previous study. In addition, since dental mesenchymal stem cells have stronger tooth differentiation capacity, the highly expressed genes in dental mesenchymal stem cells may be related to the regulation of tooth differentiation.

[0043] In the previous study, the gene expression profiles of a human non-dental stem cell, umbilical cord stem cell, and a dental stem cell, periodontal ligament stem cell, were compared by gene chip. BARX1 was found to be significantly highly expressed in the dental mesenchymal stem cell. In combination with the results of the screening of the genes of the abnormally developed tooth root and the regulatory role of BARX1 in the development of teeth, it was speculated that BARX1 might be a key gene for regulating the tooth differentiation of the stem cell in the jaw. On this basis, the further study of the research group found that overexpression of BARX1 could promote the expression of the tooth differentiation indexes DSPP and DMP1 of the apical hertwig's sheath stem cell and inhibit the expression of the osteogenic differentiation index BSP. The in vivo experiment of the repair of the dentin defect of a small pig confirmed that BARX1 had the function of promoting the apical hertwig's sheath stem cell-mediated dentin regeneration, indicating that BARX1 might be a key transcription factor for promoting the odontogenic differentiation of the apical hertwig's sheath stem cell and inhibiting the osteogenic differentiation and played an important role in the regulation of the formation of the dental mesenchymal stem cell-mediated dentin and the tooth regeneration. Then, the research group found through the tooth extraction socket replantation experiment of a rabbit that overexpression of BARX1 could significantly promote the apical hertwig's sheath stem cell-mediated dentin regeneration in the jaw, confirming that BARX1 could promote the odontogenic differentiation and regeneration function of the apical hertwig's sheath stem cell in the jaw microenvironment. However, whether BARX1 also has the function of promoting the odontogenic differentiation in the jaw in a large animal in vivo is not clear. Moreover, how BARX1 plays the functional role and whether it can improve the success rate of the biological tooth root regeneration also needs further study.

[0044] BARX1 as a transcription factor, located in the nucleus, is difficult to be directly applied in clinical treatment. Therefore, how to regulate BARX1 and its key effector genes to promote the odontogenic differentiation of apical hertial papilla stem cells and the function of tooth tissue regeneration in the jaw bone is also a problem to be solved. For its upstream regulatory mechanism, the research group found that there is an AP2a protein binding site in the 2 kb conserved sequence region of the upstream of BARX1 transcription promoter by bioinformatics analysis. At the same time, it was found that knocking out AP2a in the apical hertial papilla stem cells can up-regulate the expression of BARX1. It is speculated that BARX1 may be the downstream gene of AP2a, and its expression and function are regulated by AP2a. AP2a is a transcription factor, and its N-terminal has a transcription co-activation domain rich in proline and glutamic acid. Some studies have found that mutation of AP2a will cause mouse cleft lip and palate and facial changes, and mutation of AP2a gene in newborns will cause geniosynclitism-ocular-face syndrome and facial cleft. TFII-I and AP2a act on the promoters of some key regulatory genes of craniofacial development, indicating that AP2a plays a key role in the development of craniofacial. Previous studies have also shown that AP2a has the effect of promoting the odontogenic / bone differentiation of apical hertial papilla stem cells. The research group further found that AP2a can bind to the BARX1 promoter in apical hertial papilla stem cells by chromatin immunoprecipitation (ChIP) to inhibit the expression of BARX1. The above research results suggest that AP2a may have a regulatory effect on BARX1 in regulating the function of apical hertial papilla stem cells. However, the research group's previous functional study found that AP2a has the effect of promoting the odontogenic and bone differentiation of apical hertial papilla stem cells at the same time. Therefore, directly regulating the expression of AP2a is difficult to achieve the effect of promoting the odontogenic differentiation and inhibiting the bone differentiation of apical hertial papilla stem cells. How to promote the expression and function of BARX1 without changing the expression of AP2a is a problem we need to solve.

[0045] In recent years, small molecule polypeptide drugs have developed rapidly. After being enzymatically degraded in vivo, they are amino acids, which do not accumulate in specific organs to cause side effects. At the same time, their effects are highly targeted, with good effects. In addition, compared with active proteins, polypeptide drugs usually have only 10-100 amino acids, and their structures are simpler, making them easier to synthesize and optimize, and lower in cost, which is conducive to clinical application. Therefore, if the specific peptide segment sequence of AP2α protein binding to the BARX1 promoter region can be determined, and the small molecule polypeptide can be designed by simulating the binding site sequence, the binding site of AP2α on the BARX1 promoter can be blocked, AP2α binding to the BARX1 promoter can be blocked, the transcriptional inhibition of AP2α on BARX1 can be reduced, and BARX1 transcription can be promoted; thus, the effect of promoting the apical papilla stem cell odontogenic differentiation and inhibiting the osteogenic differentiation can be achieved. Therefore, in view of the above problems, the sequence of AP2α binding to the BARX1 promoter is designed as a DNA probe, and the specific peptide segment sequence of AP2α protein binding to the BARX1 promoter region is determined through a polypeptide microarray experiment. The DNA binding interference polypeptide that interferes with the binding of AP2α protein and BARX1 promoter is designed and synthesized according to the sequence. The function of the DNA binding interference polypeptide is further verified through in vitro and in vivo studies. In order to confirm this guess, we found that there are 11 binding sites between AP2α protein and BARX1 promoter region through protein-DNA polypeptide microarray technology:

[0046] 3 T S N G T A R L P Q L G T V G(As shown in SEQ ID No. 3)

[0047] 4 L G T V G Q S P Y T S A P P L(As shown in SEQ ID No. 4)

[0048] 6 A D F Q P P Y F P P P Y Q P I(As shown in SEQ ID No. 5)

[0049] 10-13 PQHPGWPGQRQSQESGLLHTHRGLPHQLSGLDPRRHEDLLH(As shown in SEQ ID No. 6)

[0050] 18 D Q T V I K K G P V S L S K S(As shown in SEQ ID No. 7)

[0051] 20 S A I P I N K D N L F G G V V(As shown in SEQ ID No. 8)

[0052] 22 F C S V P G R L S L L S S T S (as set forth in SEQ ID No. 9)

[0053] 26-29 GVLRRAKSKNGGRSLREKLDKIGLNLPAGRRKAANVTLLTSLVEG (as set forth in SEQ ID No. 10)

[0054] (inc. 26 FITC-(Acp)-GVLRRAKSKNGGRSLYGRKKRRQRRR (as set forth in SEQ ID No. 1)

[0055] 27 FITC-(Acp)-GGRSLREKLDKIGLNYGRKKRRQRRR (as set forth in SEQ ID No. 2)

[0056] 32 E F P A K A V A E F L N R Q H (as set forth in SEQ ID No. 11)

[0057] 34 Q V T R K N M L L A T K Q I C (as set forth in SEQ ID No. 12)

[0058] 36 FITC-(Acp)-LLAQDRSPLGNSRPNYGRKKRRQRRR (as set forth in SEQ ID No. 13)

[0059] (FITC-(Acp)- is a fluorescent tag, YGRKKRRQRRR is a cell-penetrating peptide added at the right end)

[0060] We constructed three bioactive peptides AP2a-26, AP2a-27 and AP2a-36, and designed control peptides to exclude the effects of FITC fluorescence and the penetrating peptide on cells. We screened the optimal concentration of the peptides by ALP assay and found that 10 ug / ml was the optimal concentration. Western blotting showed that AP2a-26 and AP2a-27 could specifically block the binding of AP2a to the BARX1 promoter, block the binding of the AP2a complex to the BARX1 promoter, reduce the transcriptional inhibition of AP2a on BARX1, and increase the expression of BARX1. AP2a-36 could not effectively relieve the inhibition of AP2a on BARX1. We hypothesized that blocking the transcriptional inhibition of AP2a on BARX1 could enhance the function of BARX1, promote the odontogenic differentiation of dental mesenchymal stem cells, inhibit the osteogenic differentiation of dental mesenchymal stem cells, and promote the dentin regeneration mediated by dental mesenchymal stem cells in the jaw, thereby improving the success rate of biological tooth root regeneration. We confirmed the results of the above hypothesis by in vivo tooth extraction and implantation experiments. Compared with the control group, the AP2a-26 and AP2a-27 groups showed more new tooth-like tissue formation in the tooth extraction socket. Scanning electron microscopy and HE staining showed that the AP2a-26 and AP2a-27 groups had a large number of regularly arranged dentin tubules, while the control group had irregularly arranged dentin tubules. Immunohistochemistry and quantitative results showed that the expression of DSPP was increased in the AP2a-26 and AP2a-27 groups.

[0061] Based on the mechanism study, we developed and used small molecule peptides targeting the AP2a protein and the BARX1 promoter binding region to reduce the transcriptional inhibition of AP2a on BARX1, promote BARX1 transcription, and ultimately promote the odontogenic differentiation of dental mesenchymal stem cells, inhibit the osteogenic differentiation of dental mesenchymal stem cells, and promote the dentin regeneration mediated by dental mesenchymal stem cells in the jaw, thereby improving the success rate and effect of biological tooth root regeneration.

[0062] Experimental cells:

[0063] Apical papilla stem cells (SCAPs) were obtained from orthodontic teeth or impacted third molars removed from patients (16-22 years old) with informed consent. The patients had no systemic diseases and the collected teeth had no dental and periodontal diseases. The experimental SCAPs were the 3rd-5th generation cells.

[0064] New Zealand rabbit dental pulp stem cells (rDPSCs) source: 3 kg New Zealand rabbits were anesthetized, and the dental pulp was taken out after the incisors were pulled out. After being cut into pieces, primary cell culture was carried out. The rDPSCs used in the experiment were the 3-5th generation cells.

[0065] Experimental animals:

[0066] 3 kg male New Zealand white rabbits were purchased from Beijing Fangyuanyuan Breeding Farm.

[0067] Main equipment:

[0068] Main reagents:

[0069] Statistical analysis:

[0070] SPSS 19.0 statistical software was used for statistical analysis, t test was used for comparison of two groups of measurement data, ANOVA analysis was used for comparison of multiple groups of measurement data, and P<0.05 was used as the basis for statistical difference.

[0071] Table 1 Abbreviation / Code Explanation

[0072] The polypeptide sequence of AP2 alpha and the application of regulating the function of mesenchymal stem cells provided by the application can be obtained by market purchase.

[0073] The application will be further described below in combination with examples:

[0074] Example 1 Design, synthesis and detection of polypeptide array for protein interaction research

[0075] Experimental equipment and materials

[0076] Experimental equipment: AutoSpot peptide synthesizer; Chemchemi imager

[0077] Experimental reagents and materials: PEG modified cellulose membrane, Fmoc-amino acid, DCM, amine free DMF, ethanol, Acetic anhydride, Acetic anhydride, DIPEA (Diisopropylamine), DIC (Diisopropylcarbodiimide), Bromophenol blue, Piperidine, etc. Image analysis software: TotalLab Control Centre V2009

[0078] Sample information:

[0079] Sample name Nucleic acid probe Biotin-GCCTAGGG

[0080] Polypeptide array synthesis

[0081] Activated substrate chip membrane is placed on the automatic polypeptide chip synthesizer, and Fmoc-amino acid solution is automatically transferred to the specific position of the activated membrane according to the program and reacts with the membrane. The membrane is sequentially immersed in blocking solution I and blocking solution II for side chain blocking, and the membrane is washed with DMF. The membrane is placed in a deprotection solution for removing the Fmoc protecting group at the amino terminal, and after deprotection, the membrane is washed with DMF and then dried with ethanol. Repeat the above steps until the polypeptide array is completely synthesized. After complete synthesis, remove the side chain protecting group with a specific organic reagent, wash the membrane with CH2Cl2, and then dry with ethanol, immediately use or store at -20°C for the next step experiment.

[0082] The polypeptide on the microarray is covalently bonded to the free hydroxyl group on the chip with the carboxyl group of the amino acid, and then each amino acid is added to the continuously extending polypeptide on the array until the synthesis is complete. The amino acid amino protecting group and all the amino acid side chain protecting groups on the polypeptide are removed.

[0083] Polypeptide array and recombinant protein binding reaction and antibody detection

[0084] 1. Blocking: soak the polypeptide microarray chip with anhydrous ethanol for 5 min, then add nucleic acid hybridization blocking solution, shake at room temperature for 4 hours, and wash the chip with SSC hybridization solution once for 10 min;

[0085] 2. Probe sample and polypeptide chip incubation: dilute the nucleic acid probe to 2 ug / ml with SSC hybridization solution, mix 5 ml of the diluted sample with the corresponding polypeptide microarray chip, and incubate at 4°C overnight; the control group is incubated with SSC buffer. Wash the polypeptide chip with SSC hybridization buffer 3 times x 10 min;

[0086] 3. Antibody incubation: dilute the reaction reagent Streptavidin-HRP (High Sensitivity Streptavidin-HRP (prod#21133)) with SSC hybridization buffer according to 1:10000, incubate the polypeptide microarray chip with 5 ml after dilution (1:10000), shake at room temperature for 2 hours, and wash the polypeptide chip with SSC hybridization buffer 3 times x 10 min;

[0087] 4. Color development: add ECL luminescent reagent and react for 2 min, and use Chempchemi digital imager for polypeptide chip digital imaging.

[0088] Results and analysis:

[0089] 1. Design and synthesis of polypeptide array chip

[0090] The polypeptide array film and the staining after synthesis are shown in Figure 5 (to confirm the success of synthesis, note that the staining depth does not represent the polypeptide concentration).

[0091] 2. Analysis of polypeptide array film reaction positive peptides

[0092] 2.1 Polypeptide array monoclonal antibody detection results Hybridization experiment, using the customer-provided nucleic acid probe, immunological hybridization experiment with polypeptide microarray, using Streptavidin-HRP incubation (High Sensitivity Streptavidin-HRP (prod#21133)) for hybridization reaction, color development with ECL luminescent solution, operation steps and conditions are as described above, color development for 30s, results are shown in Figure 5:

[0093] As can be seen from Figure 5, the nucleic acid probe and certain polypeptide points on the chip have obvious binding and color development, and the results are shown in Figure 5. (Chip No. 1 is the nucleic acid hybridization result, and chip No. 2 is the negative control without nucleic acid probe).

[0094] 2.2 Analysis of positive reaction points

[0095] 2.2.1 Polypeptide array and nucleic acid probe reaction results Polypeptide point gray value analysis

[0096] Using TotalLab software to analyze the color development point optical density data (see Table 2 for the original data of each point optical density value), setting the highest value of the color development point optical density value on the film as 100%, and the optical density values of the other points as the percentage value of the optical density value of the point. The polypeptide microarray chip and nucleic acid probe detection color development point data analysis chart is shown in Figure 1a, and the specific gray value data table is shown in Table 2.

[0097] In Figure 1b, the horizontal axis is the polypeptide point number, i.e. the corresponding 45 polypeptides of the array, and the vertical axis is the percentage of the point optical density value. According to experience and reference, the polypeptide chip film point optical density value exceeding 30% and the negative reaction film point optical density value below 30% are considered as positive color development points, and according to the results of Figure 1a, the following sequence (as shown in Figure 1b) has obvious differences.

[0098] Table 2

[0099] Conclusion

[0100] From the above results, it can be seen that the polypeptide array method can detect the interaction between the nucleic acid probe and the polypeptide array chip, and the interaction site exists in the polypeptide sequence listed in the above analysis table. The confirmation of the interaction site needs to be comprehensively analyzed and judged in combination with other related experimental results. The hybridization of each nucleic acid probe and the polypeptide array chip has obvious colored polypeptide points on the chip, which indicates that certain polypeptides on the polypeptide chip have obvious interaction with certain sequences in the nucleic acid probe sample. At the same time, the gray value of some continuous polypeptide points has obvious coloration, and the customer can focus on it according to the experimental requirements. The polypeptide on the site can be verified by other methods. The negative control is the polypeptide chip without nucleic acid probe directly reacted with Streptavidin-HRP, and the result shows that there is no colored point, which can confirm that Streptavidin-HRP does not react with the polypeptide chip itself.

[0101] Example 2 Obtaining, isolating and culturing of apical papilla stem cells

[0102] Under the condition that the patient is informed, the patient's orthodontic tooth or third molar is aseptically extracted under local anesthesia, and the extracted tooth is placed in a sterile centrifuge tube containing PBS with double antibodies prepared in advance. In the super-clean bench, the apical papilla tissue is scraped with a sterile blade. After repeatedly washing the removed apical papilla tissue with a large amount of PBS containing double antibodies, it is placed in a digestion solution prepared by mixing type I collagenase (3 g / L) and Dispase (4 g / L) at a ratio of 1:1 and cut into small pieces. After 40 minutes of incubation at 37°C in an incubator, 2 times the volume of medium is added to terminate the digestion, and the cells are collected into a 15 mL sterile centrifuge tube. Centrifuge at 1100 rpm / min for 6 minutes, discard the supernatant, resuspend the cell pellet by adding culture medium, mix well by blowing, inoculate in a 60 mm culture dish, and culture in a 37°C, 5% CO2 incubator. After 3 days of culture, the cell growth condition is observed under a microscope, and the fresh culture medium is replaced. When the cells grow to about 80% confluence, they are digested with 0.25% trypsin and subcultured in a 100 mm culture dish at a ratio of 1:2.

[0103] Example 3 Cell culture

[0104] Mesenchymal stem cells are cultured in MSCM medium and placed in a 37°C, 5% CO2 cell incubator. Primary cells are cultured for 3-5 passages for cell experiments. The medium is changed every three days.

[0105] Example 4 Cryopreservation and recovery of mesenchymal stem cells

[0106] (1) Cryopreservation

[0107] The cell culture medium was changed the day before freezing. The experimental supplies used, such as the gun head, pipette, centrifuge tube, etc., were sterilized in the super-clean bench for 30 minutes in advance. After washing the cells with PBS for 2 times, the cells were digested with 0.25% trypsin at 37°C for 2 minutes. After observing under an inverted microscope that the cells floated and became single cells, 3 times the volume of culture medium was added to terminate the digestion. The digested cells were suspended, mixed, and then transferred to a 15 mL centrifuge tube. Centrifugation was performed at 1100 rpm / min for 6 minutes. The supernatant in the centrifuge tube was removed, and freezing solution was added. After mixing the cells, they were aliquoted into freezing tubes and stored in a -80°C refrigerator overnight and then in a liquid nitrogen tank for long-term storage. The cell name, generation number and date were labeled on the freezing tube.

[0108] (2) Resuscitation

[0109] The experimental supplies used, such as the gun head, pipette, centrifuge tube, etc., were sterilized in the super-clean bench for 30 minutes in advance. The MSCM medium was taken out of the refrigerator and preheated in the incubator. After wearing a mask and gloves, the cells stored in liquid nitrogen were taken out and quickly placed in a 37°C water bath to melt while shaking. The melted cells were taken out of the water bath, disinfected with 75% alcohol, and the freezing tube was opened in the super-clean bench. The fine bubble suspension was aspirated and transferred to a centrifuge tube containing MSCM medium, and centrifugation was performed at 1100 rpm for 6 min. The supernatant was discarded, and the medium was added. The cells were mixed by blowing and inoculated into a culture dish, which was cultured in a 37°C, 5% CO2 incubator.

[0110] Example 5 Western Blot detection of protein expression changes

[0111] (1) Total protein extraction

[0112] After the cell culture time arrived, the experimental reagents and experimental materials were prepared in advance, and ice was prepared in advance. The culture medium in the culture dish was discarded, and 5 mL of pre-cooled PBS at 4°C was used to rinse the cells 3 times. The lysis buffer was prepared, and RIPA, PMSF and PIC were mixed in a ratio of 100:1:1. The amount added was determined according to the specific circumstances (usually 500 μL for a 10 cm culture dish), and it was placed in a 4°C refrigerator for incubation for 20 min. Every 5 min, the lysis buffer in the culture dish was shaken to cover the bottom of the dish. The cells were scraped and transferred to a 1.5 mL centrifuge tube. Centrifugation was performed at 4°C, 14000 rpm for 15 min. The supernatant was aspirated into a 1.5 mL EP tube, labeled, and stored at -80°C.

[0113] (2) Protein concentration measurement (Bradford method)

[0114] Take the protein sample stored at -80°C, quickly thaw on ice, add 200 μL 1 x Coomassie Brilliant Blue (Biobad) to the 96-well plate and add 1 μL of the protein sample (determine the amount of protein to be loaded according to the color change), mix, remove air bubbles, and load onto the machine to detect OD values. Draw a standard curve, load equal volumes and equal masses, and load 25 μg (calculate the volume of the protein, dilute to 20 μL with PBS + PMSF + PIC, and add 5 μL of 5 x loading buffer. Denature the protein at 95°C-100°C for 10 min, and store at -20°C for 10 min after denaturation.

[0115] (3) Electrophoresis

[0116] Take out the pre-gel, remove the bottom insulation strip, place the gel and back plate correctly (the font faces are all facing the experimenter), add electrophoresis buffer, and add recyclable electrophoresis liquid to the periphery according to the scale. After filling, remove the comb. Remove the pre-gel comb (move gently), add sample to each well, and add Marker to the side of the protein sample well. Add 8 μL of Marker.

[0117] (4) Transfer (do not touch water)

[0118] Take out the pre-gel, cut off the concentrated gel and the bottom part of the gel, cover the PVDF membrane on its surface (mark 1 lane position), place it on the transfer plate in the order of filter paper-PVDF membrane-gel-filter paper, remove air bubbles, cover the transfer membrane box cover, and tighten. Transfer (constant voltage 1.3V, 7min), turn off the power, take out the PVDF membrane (move quickly), wash with 1 x TBST for three times, 5min each time. Blocking: prepare blocking solution - 5% skimmed milk powder dissolved in 1 x TBST, place the PVDF membrane in the blocking solution, incubate at room temperature for 1 hour, and wash with 1 x TBST on a shaker for 4 times, 10min each time.

[0119] (5) Incubate primary and secondary antibodies

[0120] Determine the incubation antibody method according to the molecular weight difference between the internal and target proteins: if the difference is greater than 5Kda, cut the PVDF membrane to incubate the antibody; if the difference is less than 5Kda, incubate the antibody twice. Place the membrane in TBST milk containing the appropriate concentration of primary antibody (including GAPDH, HSP90, and other internal standards that may need to be incubated after cutting the membrane), and shake overnight at 4°C. Wash the membrane with 1 x TBST for three times, 5min each time. Dilute the secondary antibody at 1:2000, and shake at room temperature for 1 hour. Wash the PVDF membrane with 1 x TBST three times, 5min each time.

[0121] (6) Development

[0122] Prepare the luminescent solution in advance, mix 1:1 in darkroom. Place the PVDF membrane in the dark box, and restore the cut membrane. Drop the luminescent solution on the PVDF membrane (protein area), red light excitation for 2-3 min, and image in the BIO-RAD imaging system.

[0123] Example 6 Detection of polypeptide microarray and recombinant protein binding reaction

[0124] (1) Polypeptide chip synthesis: according to the sequence of AP2a protein, polypeptide chip was synthesized, overlapping design, a total of two arrays.

[0125] (2) Polypeptide array synthesis: the activated substrate chip membrane was placed on the automatic polypeptide chip synthesizer, and the Fmoc-amino acid solution was automatically transferred to the specific position of the activated membrane according to the program and reacted with the membrane. The membrane was immersed in blocking solution I (containing 2% (v / v) acetic anhydride in DMF (N, N-dimethylformamide, anhydrous, amine free, 99.9% solution) and blocking solution II (containing 2% (v / v) acetic anhydride, 2% (v / v) DIPEA in DMF solution) for side chain blocking, and the membrane was washed with DMF. The membrane was placed in a deprotection solution for removing the Fmoc protecting group at the amino terminal, and after deprotection, the membrane was washed with DMF and then dried with ethanol. Repeat the above steps until the polypeptide array is completely synthesized. After complete synthesis, remove the side chain protecting group with a specific organic reagent, then wash the membrane with CH2Cl2, and then dry with ethanol, immediately use or store at -20°C.

[0126] (3) Blocking: soak the polypeptide microarray chip with anhydrous ethanol for 5 min, then add nucleic acid hybridization blocking solution, shake at room temperature for 4 hours, and wash the chip with SSC hybridization solution for 10 min;

[0127] (4) Incubation of probe sample with polypeptide chip: dilute BARX1 nucleic acid probe to 2 ug / ml with SSC hybridization solution, mix 5 ml of the diluted sample with the corresponding polypeptide microarray chip, and incubate at 4°C overnight; the control group is incubated with SSC buffer. Wash the polypeptide chip with SSC hybridization buffer 3 times x 10 min;

[0128] (5) Antibody incubation: dilute the reaction reagent Streptavidin-HRP (High Sensitivity Streptavidin-HRP (prod#21133)) with SSC hybridization buffer according to 1:10000, and incubate the polypeptide microarray chip with 5 ml of the diluted nucleic acid hybridization blocking solution (1:10000), shake at room temperature for 2 hours, and wash the polypeptide chip with SSC hybridization buffer 3 times x 10 min;

[0129] (6) Color development: add ECL luminescent reagent, Chempchemi digital imager, polypeptide chip digital imaging.

[0130] (7) Chip scanning and color development point data analysis: the color development chip is scanned and imaged using the Chempchemi chemiluminescence imaging system, and the color development time is 30 s. The image is analyzed using the TotalLab image analysis software to analyze the color development point optical density value, and the "Spot Edge Average" algorithm in the software is used to calculate the optical density value of each color development point with the peripheral background value of each color development point as the reference.

[0131] Experimental Example 7 HE Staining

[0132] (1) Fixation: fresh tissues are fixed with 10% neutral formalin for 6-12 hours.

[0133] (2) Sampling: according to the experimental needs, the skull is cut into appropriate size using EXAKT312 bone sawing machine and placed in a dehydration box.

[0134] (3) Decalcification: the skull is placed in 12% EDTA decalcification solution and placed in a shaking bed for decalcification treatment, and the decalcification solution is replaced every 2-3 days until the skull becomes soft and the needle is not blocked. When there is no resistance, decalcification is completed, and the skull is washed with running water.

[0135] (4) Dehydration; wax immersion: place the dehydration box in the dehydration machine and perform dehydration in gradient alcohol. 70% alcohol for 4h-80% alcohol for 2h-90% alcohol for 2h-95% alcohol I for 1h-95% alcohol II for 1h-anhydrous ethanol I for 1h-anhydrous ethanol II for 1h-dimethylbenzene I for 1h-dimethylbenzene II for 1h-wax I for 1h-wax II for 1h-wax III for 1h.

[0136] (5) Embedding: the tissue immersed in wax is embedded in the embedding machine. First, melt the wax and place it in the embedding frame. Before the wax solidifies, remove the tissue from the dehydration box and place it in the embedding frame according to the requirements of the embedding surface. Cool on a -20°C freezing table, and after the wax solidifies, remove the wax block from the embedding frame and trim the wax block. Slice: place the trimmed wax block on a paraffin microtome and slice at a thickness of 4 μm. Float the slices on a warm water bath at about 40°C to flatten the tissue, and use a glass slide to lift the tissue. Drain the water on the tissue and place it in a 60°C oven to bake the slices.

[0137] (6) HE Staining:

[0138] Paraffin section deparaffinization to water

[0139] Xylene I 20 min

[0140] Xylene II 20 min

[0141] Anhydrous ethanol I 5 min

[0142] absolute ethanol II 5 min

[0143] 95% alcohol 2 min

[0144] 90% alcohol 2 min

[0145] 80% alcohol 2 min

[0146] 70% alcohol 2 min

[0147] distilled water wash

[0148] hematoxylin solution stain 5-10 min

[0149] tap water wash 3 times

[0150] 1% hydrochloric acid in ethanol differentiation, tap water counterstain

[0151] 0.5% eosin solution 1 min

[0152] 80% alcohol 1 min

[0153] 90% alcohol 1 min

[0154] 95% alcohol I 2 min

[0155] 95% alcohol II 2 min

[0156] absolute ethanol I 3-5 min

[0157] absolute ethanol II 3-5 min

[0158] xylene I 3-5 min

[0159] xylene II 3-5 min

[0160] neutral resin mounting

[0161] Example 8 Immunohistochemical Staining

[0162] (1) De-wax to water

[0163] (2) Staining

[0164] (3) Endogenous peroxidase blocking with hydrogen peroxide: 3% H2O2, room temperature for 20 minutes (protected from light);

[0165] (4) Distilled water wash: 5 minutes, 2 times (on a shaker);

[0166] (5) Antigen retrieval: 1% trypsin 37°C for 30 min;

[0167] (6) PBS wash: 5 minutes, 2 times (on a shaker);

[0168] (7) Goat serum blocking: remove the section from the staining jar, wipe off the water on the back of the section and the water around the tissue on the front of the section (keep the tissue moist), add normal goat or rabbit serum (same species as the secondary antibody) and incubate at 37°C for 20 minutes in a humidified chamber;

[0169] (8) Add the primary antibody: remove the serum with filter paper, do not wash, add the primary antibody directly, incubate at 37°C for 2 hours in a humidified chamber;

[0170] (9) PBS wash: 5 minutes, 2 times (on a shaker);

[0171] (10) Add the secondary antibody labeled with horseradish peroxidase, incubate at room temperature for 30 minutes, PBS wash, 5 minutes, 3 times (on a shaker);

[0172] (11) Develop with DAB working solution, wash with distilled water;

[0173] (12) Hematoxylin stain the nucleus;

[0174] (13) Dehydrate with gradient alcohol, clear with xylene, mount with neutral resin, observe under a microscope and take pictures.

[0175] Effect Example 1: Study on the regulatory mechanism of the influence of AP2a / BARX1 on the osteogenic and odontogenic differentiation ability of dental mesenchymal stem cells

[0176] 1.1 There are 11 binding sites between AP2a protein and BARX1 promoter nucleic acid, and AP2a-26 and AP2a-27 can effectively block the inhibitory effect of AP2a protein on BARX1

[0177] In order to further study the functional regulation mechanism of AP2a and BARX1 on dental mesenchymal stem cells, the research group used polypeptide microarray technology to find the binding sites and sequences of AP2a protein and BARX1 promoter nucleic acid. Polypeptide microarray technology found that there were 11 positive binding sites between AP2a protein and BARX1 promoter nucleic acid (Figure 1a). According to the binding site analysis (Figure 1b) and sequence design, AP2a-26, AP2a-27 and AP2a-36 were synthesized.

[0178] 26 FITC-(Acp)-GVLRRAKSKNGGRSLYGRKKRRQRRR (as shown in SEQ ID No. 1)

[0179] 27 FITC-(Acp)-GGRSLREKLDKIGLNYGRKKRRQRRR (as shown in SEQ ID No. 2)

[0180] 36 FITC-(Acp)-LLAQDRSPLGNSRPNYGRKKRRQRRR (as set forth in SEQ ID No. 13)

[0181] Western blot results and gray value analysis found that only AP2a-26, AP2a-27 in AP2a-26, AP2a-27, AP2a-36 can open the binding of AP2a protein to the DNA promoter region of BARX1, block the inhibition of AP2 protein to BARX1, and make the expression of BARX1 increase (Fig. 1c-d).

[0182] Table 3 Data of Fig. 1d

[0183] 1.2 Polypeptides AP2a-26 and AP2a-27 increase the binding of BARX1 / OSX protein complex in apical herticle stem cells

[0184] Previous studies have shown that BARX1 can form a protein complex with OSX to regulate the odontogenic differentiation function of apical herticle stem cells. Therefore, we further explored the effect of polypeptides AP2a-26 and AP2a-27 on the BARX1 / OSX protein complex in apical herticle stem cells. Western blot results showed that the expression of BARX1 and OSX increased after adding AP2a-26 and AP2a-27 in apical herticle stem cells, and Co-IP results showed that AP2a-26 and AP2a-27 increased the binding of BARX1 / OSX protein complex in apical herticle stem cells (Fig. 2).

[0185] 1.3 Polypeptides AP2a-26 and AP2a-27 can increase the binding of BARX1 / OSX protein complex in New Zealand rabbit dental pulp stem cells

[0186] We also explored whether polypeptides AP2a-26 and AP2a-27 can increase the expression of BARX1 / OSX protein complex in New Zealand rabbit dental pulp stem cells. Western blot results showed that the expression of BARX1 and OSX increased after adding AP2a-26 and AP2a-27 in New Zealand rabbit dental pulp stem cells, and the expression of osteogenic marker gene BSP decreased, indicating that AP2a-26 and AP2a-27 inhibited the osteogenic differentiation function of rabbit dental pulp stem cells; Co-IP results showed that AP2a-26 and AP2a-27 increased the binding of BARX1 / OSX protein complex in New Zealand rabbit dental pulp stem cells (Fig. 3).

[0187] Example 2 In vivo study of AP2a / BARX1 on dentin regeneration in New Zealand white rabbit extraction socket

[0188] 2.1 Establishment of New Zealand white rabbit tooth extraction socket model

[0189] New Zealand white rabbits were anesthetized by intramuscular injection, and 6 rabbits were randomly allocated in each group. The right incisors were extracted from the maxilla and mandible, and then the tissue debris was completely removed. During the extraction process, we completely extracted the teeth, and then used a curette to completely remove the residual periodontal membrane and apical tooth papilla in the extraction socket, a total of 30 extraction socket models were generated, which were randomly divided into 5 groups: Mock group, Matrigel group, SCAP / Control group, SCAP / AP2a-26 group and SCAP / AP2a-27 group.

[0190] 2.2 AP2a-26 and AP2a-27 regenerate and repair New Zealand white rabbit tooth extraction socket

[0191] After 12 hours of AP2a-26 and AP2a-27 pretreatment of SCAPs, 100 μL of SCAPs (1 × 10 6 ) and 150 μL of Matrigel matrix mixture were transplanted into the extraction socket (SCAP / Control group, SCAP / AP2a-26 group and SCAP / AP2a-27 group), and the extraction socket was closed with 4-0 suture line. The animals were sacrificed 12 weeks after the operation, and samples were obtained and subjected to micro-CT tomography (80 kV, 2 s, Siemens Inveon, Munich, Germany). Micro-CT showed that among the 30 extraction sockets: no obvious tooth-like tissue was found in the Mock group, no obvious tooth-like tissue was found in the Matrigel group, 1 extraction socket had tooth-like tissue in the Control group, tooth-like tissue was found in all extraction sockets in the AP2a-26 group, and tooth-like tissue was found in 4 extraction sockets in the AP2a-27 group (2 died during feeding) (Fig. 4a-b); scanning electron microscopy and HE staining results showed that a large number of regularly arranged dentin tubules were observed in the AP2a-26 and AP2a-27 groups, while the dentin tubules in the Control group were arranged irregularly; immunohistochemistry and quantification results showed that the expression of DSPP increased in the AP2a-26 and AP2a-27 groups (Fig. 4c-f).

[0192] Table 4 Data of Fig. 4b

[0193] Among them, “-”: 2 experimental animals died, therefore, 4 groups of data are missing.

[0194] Table 5 Data of Fig. 4f

[0195] The polypeptide sequence based on the AP2α sequence designed by the application and the application of regulating the functions of the dental mesenchymal stem cells are described in detail. The principle and implementation mode of the application are described by using specific examples, and the above examples are only used to help understand the method of the application and its core idea. It should be pointed out that the person skilled in the art can make some improvements and modifications to the application without departing from the principle of the application, and these improvements and modifications also fall within the protection scope of the claims of the application.

Claims

1. A polypeptide, characterized in that, which has: (I) an amino acid sequence as shown in any one of SEQ ID No. 3-13; (II) an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids of the amino acid sequence as described in (I), and an amino acid sequence which is functionally identical to the amino acid sequence as described in (I); or (III) an amino acid sequence having 90% or more identity to the amino acid sequence as described in (I) or (II).

2. Use of the polypeptide as claimed in claim 1 as a binding site of AP2α protein to the BARX1 promoter region in the preparation of a bioactive peptide interfering with the binding of AP2α protein to the BARX1 promoter.

3. A biologically active peptide characterized in that, which has: (I) an amino acid sequence as shown in SEQ ID No. 1 or 2; (II) an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids of the amino acid sequence as described in (I), and an amino acid sequence which is functionally identical to the amino acid sequence as described in (I); or (III) an amino acid sequence having 90% or more identity to the amino acid sequence as described in (I) or (II).

4. Use of the bioactive peptide as claimed in claim 3 in the preparation of a reagent or a drug for any one of: (I) specifically blocking the binding site of AP2α on the BARX1 promoter; (II) blocking the binding of AP2α complex to the BARX1 promoter; (III) reducing the transcriptional inhibition of AP2α on BARX1; and / or (IV) promoting the transcription of BARX1.

5. Use of the bioactive peptide as claimed in claim 3 in the preparation of a reagent or a drug for promoting the increase of the binding of BARX1 / OSX protein complex in odontogenic mesenchymal stem cells.

6. Use of the bioactive peptide as claimed in claim 3 in the preparation of a reagent or a drug for promoting the odontogenic differentiation of odontogenic mesenchymal stem cells.

7. Use of the bioactive peptide as claimed in claim 3 in the preparation of a reagent or a drug for odontogenic mesenchymal stem cell-mediated dentin regeneration and / or biological root regeneration in the jaw.

8. Use of the bioactive peptide as claimed in claim 3 in the preparation of a reagent or a drug for improving the success rate and / or effect of biological root regeneration.

9. Use according to any one of claims 5 to 8, wherein the compound is ###0002### The odontogenic mesenchymal stem cells include apical papilla stem cells or dental pulp stem cells.

10. An agent or medicament, characterized in that, The bioactive peptide as claimed in claim 3 is included.

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