SFRP2 functional polypeptide and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an SFRP2 functional polypeptide and application thereof. The invention provides an SFRP2 functional polypeptide and application thereof. The SFRP2 functional polypeptide (SFRP2-PP96) can be used for reducing the proportion of Th17 subgroups in CD4 + T cells and increasing the proportion of Treg subgroups; the differentiation of CD4 + T cells in induction to the Th17 subgroup is inhibited, and the differentiation to the Treg subgroup is promoted; the experimental colitis of mice is inhibited; the proliferation of Th17 in colon tissues is inhibited, and the proliferation of Treg is promoted; the expression of pro-inflammatory protein in the inflammatory colon tissue is inhibited, and the expression of anti-inflammatory protein is promoted; colitis recovery is promoted by adjusting the proportion of intestinal flora; the treatment effect on experimental periodontitis is promoted. And the polypeptide preparation has characteristics superior to those of the traditional full-length protein, and is convenient for subsequent clinical application and popularization.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on December 22, 2023, with application number 202311786953.0 and invention name “SFRP2 functional polypeptide and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to SFRP2 functional polypeptides and applications thereof. Background Art

[0003] Periodontitis is a complex chronic infectious disease caused by multiple etiologies and contributing factors, which usually appears in early adulthood. Persistent inflammation of the periodontal tissue can cause deepening of periodontal pockets, periodontal suppuration, periodontal bone loss, and loosening and falling of teeth. The repair of periodontal bone defects is a major challenge in the field of regeneration. Mild to moderate periodontitis is generally controlled by non-surgical treatments and oral hygiene education. Severe periodontitis requires surgical treatment, but traditional treatments such as guided bone regeneration and guided tissue regeneration have limited and unstable regeneration effects and cannot achieve complete regeneration of periodontal tissues. In addition, there are complex immune responses in the periodontal tissues of periodontitis, including innate and adaptive immune responses, which are aimed at controlling the invasion of pathogens. However, dysregulation of the innate and adaptive immune systems may play an important role in the etiology of periodontitis. Cytokines produced by the immune response regulate the interactions and cell networks between macrophages, T cells, B cells, epithelial cells, and other cell types. Proinflammatory cytokines activate immune defense while also activating osteoclasts and matrix metalloproteinases (collagenases); anti-inflammatory cytokines participate in the maturation, proliferation, and isotype switching of B cells, thereby inhibiting the progression of periodontitis. Severe periodontitis may not be cured by traditional therapies, and immunotherapy can serve as an adjuvant or alternative to traditional therapies. Immunotherapy has been shown to potentially regulate dysregulated immunity in autoimmune and inflammatory diseases. Current treatments for periodontitis focus on periodontal cleaning and scaling, or sometimes require combined antibiotic treatment to remove biofilms. However, these therapies often fail to completely remove periodontal pathogens, and their continued presence continues to stimulate the host's immune response. In turn, the host's dysregulated immune response leads to further spread of periodontal pathogens.

[0004] In the immune response of periodontitis, CD4 + T cells play an important role. Th17 cells are CD4 +A unique lineage of T cells promotes the progression of periodontitis through the secretion of the key cytokine IL-17. Treg cells are regulated by the transcription factor Foxp3, which induces the differentiation of Treg cells and downregulates the differentiation of Th17 cells through STAT6. The dynamic balance between Th17 and Treg is an important regulatory factor in inflammation. So far, the cause of Th17 / Treg imbalance during periodontitis has not been fully elucidated.

[0005] The Wnt / β-catenin signaling pathway plays a key role in tooth development and differentiation. Currently, there is still controversy over the role of the Wnt / β-catenin signaling pathway in periodontitis.

[0006] Secreted frizzled-related protein 2 (SFRP2), as an antagonist of the Wnt / β-catenin signaling pathway, has many advantages, such as regulating the osteogenic / odontogenic differentiation of mesenchymal stem cells, promoting tissue repair and regeneration, and regulating immune balance. However, traditional recombinant proteins have low purity, low yield, long production cycle, high production cost, and cannot introduce non-natural amino acids or amidate at their ends, which limits their further clinical transformation and application. Summary of the invention

[0007] In view of this, the present invention provides SFRP2 functional polypeptides and applications thereof.

[0008] The present invention provides SFRP2 functional polypeptide and its application. SFRP2 functional polypeptide (SFRP2-PP96) can reduce the proportion of Th17 subgroup in CD4+T cells and increase the proportion of Treg subgroup; inhibit the differentiation of induced CD4+T cells to Th17 subgroup and promote differentiation to Treg subgroup; inhibit experimental colitis in mice; inhibit Th17 proliferation in colon tissue and promote Treg proliferation; inhibit the expression of pro-inflammatory proteins in inflamed colon tissue and promote the expression of anti-inflammatory proteins; promote the recovery of colitis by regulating the proportion of intestinal flora; and promote the therapeutic effect of experimental periodontitis. And it is a polypeptide preparation, which has the characteristics superior to traditional full-length proteins, and is convenient for subsequent clinical application and promotion.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a SFRP2 polypeptide having:

[0011] (1), the amino acid sequence shown in SEQ ID No. 1 or 2; or

[0012] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0013] (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (I).

[0014] In some embodiments of the present invention, the SFRP2 polypeptide has:

[0015] (1), the amino acid sequence shown in SEQ ID No. 2; or

[0016] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0017] (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (I).

[0018] The present invention also provides the use of the SFRP2 polypeptide in any of the following items:

[0019] (1) regulating the osteogenic and / or odontogenic differentiation of mesenchymal stem cells; and / or

[0020] (2) Promote tissue repair and regeneration and / or regulate immune balance.

[0021] In some specific embodiments of the present invention, the mesenchymal stem cells include but are not limited to apical dental papilla stem cells.

[0022] In some embodiments of the invention, the tissue comprises periodontal tissue.

[0023] In some specific embodiments of the present invention, the SFRP2 polypeptide is used in the preparation of a medicament for preventing and / or treating periodontitis.

[0024] In some embodiments of the present invention, the prevention and / or treatment of periodontitis comprises reducing CD4 + Increased proportion of Th17 subsets and / or CD4 + The proportion of Treg subsets in T cells;

[0025] The method for preventing and / or treating periodontitis further comprises inhibiting CD4 + T cells differentiate into Th17 subsets and / or promote CD4 + T cells differentiate into Treg subsets.

[0026] In some specific embodiments of the present invention, the prevention and / or treatment of periodontitis further comprises:

[0027] (I) Promote the migration ability of DPSCs;

[0028] (II), promoting the chemotactic ability of DPSCs;

[0029] (III) promoting the odontogenic differentiation ability of DPSCs in vitro;

[0030] (IV), rescue of odontogenic differentiation ability of SFRP2shDPSCs;

[0031] (V) Promote DPSCs-mediated tooth regeneration in the jaw;

[0032] (VI) Inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a.

[0033] Based on the above research, the present invention also provides the use of the SFRP2 polypeptide in the preparation of a drug for preventing and / or treating colitis.

[0034] In some embodiments of the present invention, the prevention and / or treatment of colitis comprises inhibiting CD4 + Proliferation of Th17 subsets in T cells and / or promotion of CD4 + Proliferation of Treg subset in T cells.

[0035] In some specific embodiments of the present invention, the prevention and / or treatment of colitis comprises inhibiting the expression of pro-inflammatory proteins and / or promoting the expression of anti-inflammatory proteins.

[0036] In some specific embodiments of the present invention, the pro-inflammatory proteins include IL-17A and IL-22; the anti-inflammatory proteins include IL-10 and TGF-β1.

[0037] In some specific embodiments of the present invention, the prevention and / or treatment of colitis comprises regulating the ratio of intestinal flora.

[0038] In some specific embodiments of the present invention, regulating the proportion of intestinal flora includes reducing the proportion of Firmicutes and / or Bacteroidetes.

[0039] In some specific embodiments of the present invention, regulating the proportion of intestinal flora includes increasing the proportion of Muribaculaceae bacteria and / or Dunaliella.

[0040] The present invention also provides a drug comprising the SFRP2 polypeptide.

[0041] The present invention also provides a drug combination, comprising the drug and any other effective ingredients.

[0042] The present invention provides SFRP2 functional polypeptide and its application. The SFRP2 polypeptide of the present invention has high purity, shortens the production cycle, can be modified with amino acids at both ends of the polypeptide to improve solubility, and its metabolites are amino acids, which are non-toxic. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0044] Figure 1 The gray value bar graph of the peptide chip in effect example 1 is shown;

[0045] Figure 2 The peptide chip array results in Example 1 are shown;

[0046] Figure 3 The purity, molecular weight, and solubility information of the control peptide ConPP and the functional peptide SFRP2-PP96 in Example 1 are shown;

[0047] Figure 4 The in vitro osteogenic induction results of different peptide groups in the test examples are shown;

[0048] Figure 5 The results of the in vitro migration ability of human dental pulp stem cells by SFRP2 functional polypeptide in the test example are shown;

[0049] Figure 6 The results of the in vitro chemotactic ability of SFRP2 functional polypeptide on human dental pulp stem cells in the test examples are shown;

[0050] Figure 7 The results of the detection of Th17 and Treg subpopulations by the SFRP2 functional polypeptide in Example 2 are shown;

[0051] Figure 8 The results of the detection of Th17 and Treg subsets by the SFRP2 functional polypeptide under Th17 polarization conditions in Example 3 are shown;

[0052] Fig. 9 The results of the detection of Th17 and Treg subsets by the SFRP2 functional polypeptide under Treg polarization conditions in Example 3 are shown;

[0053] Fig.10 The results of the treatment of experimental colitis in mice by the SFRP2 functional polypeptide in Example 4 are shown;

[0054] Fig.11 The results of the detection of Th17 and Treg subpopulations in mesenteric lymph nodes and colon tissues by the SFRP2 functional polypeptide in Example 5 are shown;

[0055] Fig.12The detection of inflammation-related protein chip in colon tissue by SFRP2 functional polypeptide in Example 6 is shown;

[0056] Fig.13 The effect of the SFRP2 functional polypeptide in Example 7 on regulating intestinal flora is shown;

[0057] Fig.14 3D stereoscopic imaging showing the therapeutic effect of SFRP2 functional polypeptide on experimental periodontitis in mice in Example 8;

[0058] Fig.15 The results of the in vitro odontogenic differentiation of human dental pulp stem cells by the SFRP2 functional polypeptide in Example 9 are shown;

[0059] Fig.16 The SFRP2 functional polypeptide in Example 9 partially rescues the in vitro odontogenic differentiation ability of SFRP2sh dental pulp stem cells;

[0060] Fig.17 The method for establishing the tooth regeneration model in the rabbit jaw in Example 10 is shown;

[0061] Fig.18 The SFRP2 functional polypeptide in Example 10 promotes the regeneration of teeth in the rabbit jaw mediated by dental pulp stem cells;

[0062] Fig.19 The SFRP2 functional polypeptide in Example 11 inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a. DETAILED DESCRIPTION

[0063] The present invention discloses SFRP2 functional polypeptides and their applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0064] The chemical synthesis technology of peptides is now increasingly mature, with low R&D and synthesis costs, and peptide products are easy to separate from impurities, so the purity is high. In addition, peptides are easy to introduce non-natural amino acids for modification during the production process. Since peptides are mainly degraded into amino acids through proteolysis and cleared by kidney filtration, the metabolites are non-toxic.

[0065] The SFRP2 polypeptide of the present invention has high purity and shortens the production cycle. Amino acid modification can be performed at both ends of the polypeptide to improve solubility. The metabolites are amino acids and the products are non-toxic.

[0066] After initially clarifying the regulatory effect of full-length SFRP2 protein on Th17 / Treg balance and its therapeutic effect on periodontitis, we found that the Fz region of SFRP2 protein can bind to Wnt ligands through website query and literature search. How to find the binding fragments of SFRP2 and Wnt ligands for more accurate subsequent research and clinical transformation applications needs to be solved urgently. With the help of peptide microarray analysis, we reported the key interaction fragment site information of SFRP2 binding to Wnt ligands for the first time. The peptide microarray results showed that there were two sites with strong binding, namely sites 7-8 (named SFRP2-PP95, amino acid sequence: LQLCHGIEYQNMRLPNL, SEQ ID No.1) and sites 49-54 (named SFRP2-PP96, amino acid sequence: AFGFPWPDMLECDRFPQDNDLCIPL, SEQ ID No.2). Subsequently, we synthesized these two peptides and used ConPP (amino acid sequence: EEEACDQQPQEEEEKDEEGE, SEQ ID No.3) for control. SFRP2 functional peptides can block the Wnt signaling pathway by binding to the putative Wnt ligand of Frizzled receptors. In order to screen the optimal concentration of functional peptides acting on cells, based on the results of in vitro osteogenic differentiation experiments of human dental pulp stem cells (DPSCs), the site 49-54 (i.e., SFRP2-PP96) peptide was selected for subsequent experimental studies.

[0067] SFRP2 functional polypeptide (SFRP2-PP96) can achieve the same immune regulation and periodontitis treatment effects as recombinant SFRP2 protein, but because it is a polypeptide preparation, it has superior properties to traditional full-length protein, which facilitates subsequent clinical application and promotion.

[0068] The SFRP2 functional polypeptide provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0069] The present invention will be further described below in conjunction with embodiments:

[0070] Preparation Example Peptide Array Chip Synthesis:

[0071] 1. Peptide array chip synthesis:

[0072] According to the sequence of SFRP2 protein (the sequence number of SFRP2 protein is Q96HF1), a peptide array was synthesized, and two synthetic peptide array chips were prepared according to the overlapping design and peptide array chip SOP (Standard Operating Procedure):

[0073] (1) The activated matrix chip membrane was placed on a fully automatic peptide chip synthesizer (Aurora Group Company, VERSA110), and the Fmoc-amino acid solution (Chengdu Chengnuo Biotechnology Co., Ltd., 20 kinds) was automatically transferred to a specific position on the activated membrane according to the program to react with the membrane;

[0074] (2) After each layer is synthesized, the membrane is immersed in blocking solution I (DMF (N,N-Dimethylformamide, anhydrous, amine free, 99.9%, Alfa-Asia Product No.: 043465) solution containing 2% (v / v) acetic anhydride (Sinopharm Chemical Reagent Co., Ltd., Product No.: 10000317)) and blocking solution II (DMF solution containing 2% (v / v) acetic anhydride and 2% (v / v) DIPEA (N,N-Diisopropylethylamine, Aldrich Product No.: 550043)) in sequence for side chain blocking, and then the membrane is washed with DMF (N,N-dimethylformamide, Sinopharm Chemical Reagent Co., Ltd., Product No.: CST12767S) for 5 times, each time for 3 minutes;

[0075] (3) The membrane was placed in a piperidine deprotection solution (DMF solution containing 20% ​​(v / v) piperidine (Sinopharm Chemical Reagent Co., Ltd., Catalog No.: 80104216)) to remove the Fmoc-protecting group at the amino terminal. After deprotection, the membrane was washed with DMF 5 times for 3 minutes each time, and then washed with ethanol (Sinopharm Chemical Reagent Co., Ltd., Catalog No.: 100092008) 5 times for 3 minutes each time, and dried. The above steps were repeated until the peptide array was fully synthesized;

[0076] (4) After the chip is fully synthesized, the side chain protecting groups are removed with piperidine, and the membrane is washed with DMF 5 times, 3 minutes each time. Then the membrane is washed with CH2Cl2 (dichloromethane, Sinopharm Chemical Reagent Co., Ltd., catalog number: 8004731916) 5 times, 3 minutes each time, to remove all the protecting groups on the peptide step by step; finally, the membrane is washed with CH2Cl2, washed with DMF 5 times, 3 minutes each time, and finally washed with ethanol, washed with DMF 5 times, 3 minutes each time, and dried naturally. The completed peptide array chip is used immediately or stored at -20℃ / -80℃.

[0077] Example 1 Peptide array chip and serum immune response detection

[0078] (1) Peptide array chip sealing:

[0079] After the peptide array chip is activated, add the blocking solution and shake and block at 20℃±5℃ for 4 hours;

[0080] (2) Incubation of the reaction protein sample with the peptide array chip: The tested reaction protein Wnt3a was diluted with blocking solution and incubated with the peptide array chip. The experimental group used 10 mL of 5 μg / mL Wnt3a recombinant protein reaction solution, and the control group chip used blocking solution. Shake overnight at 4°C, remove the membrane, and wash the membrane with TBST (Tris Buffered Saline with Tween 20) 5 times, 10 minutes each time;

[0081] (3) Incubation of peptide array chip with primary antibody: dilute the primary antibody (Anti-Wnt3a antibody) at 1:1000, incubate with 10 mL each of the experimental group and the control group, shake at 20°C±5°C for 2 h, and wash the membrane 5 times, 10 min each time;

[0082] (4) Incubation of peptide array chip with HRP-labeled secondary antibody: After HRP-labeled secondary antibody was diluted at 1:10000, 10 mL of the experimental group and the control group were incubated with each, shaken at 20°C±5°C for 2 h, and the membrane was washed 5 times, each for 10 min;

[0083] (5) Color development: ECL luminescent reagent (Pierce ECL; Thermo Fisher Scientific; #32109, USA) was added, and digital imaging was performed using a Chempchemi digital imager (VILBERFUSION FX7 Spectra, Vilber, France).

[0084] Effect Example 1

[0085] 1. Synthesis of peptides

[0086] According to the array results, the sequences corresponding to the obvious color spots were selected for peptide synthesis and subsequent functional verification.

[0087] 2. The array results are shown in Table 1. Figure 1 and Figure 2 .

[0088] Table 1

[0089]

[0090]

[0091] from Figure 1 , 2 The data can be analyzed to find 7-8 points on the peptide array (amino acid sequence:

[0092] LQLCHGIEYQNMRLPNL, SEQ ID No. 1) and 49-54 (amino acid sequence:

[0093] AFGFPWPDMLECDRFPQDNDLCIPL, SEQ ID No. 2) has a color development effect.

[0094] Subsequently, a peptide concentration of 10 μg / mL was used for in vivo and in vitro experimental studies.

[0095] 3. The purity, molecular weight and solubility information of the control peptide ConPP and the functional peptide SFRP2-PP96 can be found in Figure 3 The control polypeptide ConPP and SFRP2-PP96 are both polypeptides produced in the present invention.

[0096] As shown in the figure, the control polypeptide ConPP has a purity of 98.5%, a molecular weight of 2380.31, and can be dissolved in 100% H2O at a concentration of 1 mg / mL.

[0097] The functional polypeptide SFRP2-PP96 has a purity of 98.2%, a molecular weight of 2940.37, and can be dissolved in 100% DMSO at a concentration of 1 mg / mL.

[0098] Experimental example on the selection of SFRP2-PP95 and SFRP2-PP96

[0099] Fourth-generation (P4) DPSCs were used for in vitro osteogenic induction: trypsin (Gibco) digestion and centrifugation were performed, and the cells were resuspended in medium containing fetal bovine serum (FBS, Gibco) and counted with a hemocytometer to a cell density of 1 × 10 in a 6-well plate. 5 / well. When the cells grow to a density of 70%, the culture medium is sucked off, and the cells are washed with phosphate buffered saline (PBS, Biosharp) and replaced with osteogenic induction medium. The osteogenic induction medium is replaced every 2 to 3 days. Among them, the DPSCs group was used as the control group, the DPSCs+ConPP group maintained 10 μL / mL ConPP in the culture medium during osteogenic induction, the DPSCs+SFRP2-PP95 group maintained 10 μL / mL SFRP2-PP95 in the culture medium during osteogenic induction, and the DPSCs+SFRP2-PP96 group maintained 10 μL / mL SFRP2-PP96 in the culture medium during osteogenic induction. After 14 days of continuous induction, the culture medium was discarded, washed three times with PBS, and the cells were fixed with tissue fixative (Biyuntian) at room temperature for 30 minutes, washed three times with PBS, and 1 mL of 2% Alizarin Red Solution (Sigma) was dripped into each well. After staining for 30 minutes, the dye solution was sucked off, and the cells were rinsed with PBS three times. Whether mineralized nodules appeared was observed under an inverted microscope and pictures were collected.

[0100] 10% CPC aqueous solution (cetylpyridinium chloride, self-prepared) was added to the well plate to dissolve Ca2+ ions, 1 mL per well, and incubated at 20°C ± 5°C for 30 minutes. The solution was diluted 1:10, and the absorbance value (OD value) was measured at 562 wavelength in an ELISA reader. The experimental data were processed using statistical analysis software SPSS22.0 and Prism 7.0, and one-way ANOVA was used for statistical analysis. The data were expressed as mean ± SEM, and statistically significant when P < 0.05.

[0101] Fourth-generation (P4) DPSCs were used for in vitro osteogenic induction: trypsin (Gibco) digestion and centrifugation were performed, and the cells were resuspended in medium containing fetal bovine serum (FBS, Gibco) and counted with a hemocytometer to a cell density of 1 × 10 in a 6-well plate. 5 / well. When the cells grow to a density of 70%, the culture medium is aspirated, washed with phosphate buffered saline (PBS, Biosharp), and replaced with osteogenic induction medium. The osteogenic induction medium is replaced every 2 to 3 days. The grouping is the same as above. After 7 consecutive days of induction, the culture medium is discarded, washed three times with PBS, 600μL lysis buffer (Biyuntian) is added, and incubated at 37°C for 15 minutes. Scrape the cells and transfer them into a 1.5mL EP tube, centrifuge at 14000rpm and 4°C for 10 minutes. Add 50μL Alkaline buffer sol and 50μL Stock Substrate sol to each well of the 96-well plate, add 10μL of the lysed cell sample and mix well, and incubate at 37°C for 15 minutes. Detect the absorbance of the well plate at 405nm.

[0102] The results showed that the DPSCs+SFRP2-PP95 and DFSCs+SFRP2-PP96 groups showed stronger mineralization ability than the control group and the control peptide group ( Figure 4 A). The results of calcium ion quantitative detection showed that the calcium ion concentrations in the DPSCs+SFRP2-PP95 and DFSCs+SFRP2-PP96 groups were significantly higher than those in the control group and the control peptide group, and the effect of SFRP2-PP96 was more significant than that of SFRP2-PP95 ( Figure 4 B, Table 2). The ALP activity results showed that the SFRP2-PP96 group showed higher ALP activity, while the SFRP2-PP95 group did not show statistical differences compared with the control group and the control peptide group ( Figure 4 C shown, Table 2).

[0103] Table 2

[0104]

[0105] Cell scratch assay: DPSCs were cultured at 5 × 104 The cells were inoculated at a density of 100 μg / well in a 6-well plate and grown to 90% confluence. After 24 hours of serum-free culture, a 10 μL pipette tip was used to make cross scratches along the aperture, and then fresh culture medium was added. The scratches at 0 h, 24 h, and 48 h were observed under a microscope at the same viewing angle to detect the degree of healing of the scratches. ImageJ1.49v software was used to detect the blank area, and the degree of healing of the scratches was determined according to the percentage of blank area in each group.

[0106] Cell Transwell experiment: Prepare a 0.1% collagen solution at a concentration of 6-10 ug / cm 2 Coat the Transwell chamber with a concentration of 100 μg / mL and bind at 37°C for several hours. Remove excess liquid from the coated surface and dry overnight. Add serum-free culture medium containing BSA and place at 37°C for 30 minutes, then aspirate the residual liquid in the culture plate. Take the cell density to 2×10 5 100 μL of DPSCs cell suspension with a concentration of 100 μL / mL was added to the upper chamber of the Transwell. DPSCs were cultured without serum for 24 h before preparing the cell suspension. 600 μL of α-MEM medium containing 5% fetal bovine serum was added to the lower chamber of the 24-well plate. The culture was routinely cultured for 6 h. The Transwell chamber was removed, the culture medium in the well was discarded, and the well was washed twice with calcium-free PBS. The well was fixed with methanol for 30 minutes, and the chamber was properly air-dried. 0.1% crystal violet was used for 20 min. The unmigrated cells in the upper layer were gently wiped off with a cotton swab and washed three times with PBS. The cells were observed in 5 fields of view under a 400x microscope and counted.

[0107] The results showed that at 24, 48 and 72 h, the DPSCs+SFRP2-PP95 group showed stronger migration ability than the control group and the control peptide group ( Figure 5 A, B, C, D shown in Table 3); at 24 and 72 h, the DPSCs+SFRP2-PP96 group showed stronger migration ability than the control group and the control peptide group ( Figure 5 In addition, at 24 and 48 h, the DPSCs+SFRP2-PP95 group showed stronger chemotaxis than the control group and the control peptide group ( Figure 6 A, B, and C shown in Table 4); at 24 h, the DPSCs+SFRP2-PP96 group showed stronger chemotaxis than the control group and the control peptide group ( Figure 6 A, B shown).

[0108] Table 3

[0109]

[0110] Table 4

[0111]

[0112] The above results show that SFRP2-PP96 peptide may have stronger biological functions, such as promoting osteogenic differentiation of DPSCs. Therefore, SFRP2-PP96 was selected as the SFRP2 functional peptide in subsequent experiments.

[0113] Example 2 SFRP2-PP96 reduces the proportion of Th17 subsets and increases the proportion of Treg subsets in CD4+T cells

[0114] In order to study the effect of SFRP2 functional polypeptide on CD4+T cell subsets, especially Th17 and Treg subsets, flow cytometry and Real-time RT-PCR techniques were used for detection.

[0115] First, CD4 + T cells, in CD4 + T cells were cultured in an environment with 10 μg / mL of ConPP (control peptide) and SFRP2-PP96 for 3 days, and the cells were subjected to CD4 surface markers and IL-17A intracellular staining, and flow cytometry was used to detect Th17 subpopulation staining; or cells were subjected to CD4 and CD25 surface markers and Foxp3 nuclear transcription factor staining, and flow cytometry was used to detect Treg subpopulation staining. After the machine test, Flowjo 10.4.0 software was used to perform lymphocyte gating, remove adhesion cells, and remove dead cells in turn, and then analyze CD4 + IL-17A + Th17 subset ratio and CD4 + CD25 + Foxp3 + The results showed that compared with the blank control group, the CD4 + There was no significant change in the proportion of Th17 subset and Treg subset in T cells (P>0.05, Figure 7 AB, DE shown), while CD4 + The proportion of Th17 subset in T cells decreased (P<0.05, Figure 7 AB shown in Table 5), the proportion of Treg subsets increased (P < 0.05, Figure 7 DE shown, Table 5)).

[0116] At the same time, total RNA was extracted from the remaining duplicate wells of the same batch of cells, and the relative expression levels of mRNA of Th17 key transcription factor RORγt and Treg key transcription factor Foxp3 were detected by Real-time RT-PCR technology. The results showed that compared with the blank control group, CD4 + There was no significant change in the relative mRNA expression of RORγt and Foxp3 in T cells (P>0.05, Figure 7 C and F), while CD4 + The relative expression of RORγt mRNA in T cells was significantly decreased (P<0.01, Figure 7 C, Table 6), the relative expression of Foxp3 mRNA was significantly increased (P < 0.01, Figure 7 F shown, Table 6).

[0117] The above results suggest that under in vitro conditions, SFRP2 functional peptide SFRP2-PP96 reduces CD4 + The proportion of Th17 subpopulation in T cells was increased, and the proportion of Treg subpopulation was increased, while the control polypeptide had no such effect.

[0118] Table 5

[0119]

[0120] Table 6

[0121]

[0122] Example 3 SFRP2-PP96 inhibits the differentiation of induced CD4+ T cells into Th17 subsets and promotes differentiation into Treg subsets

[0123] Similarly, CD4 + T cells, in CD4 +2ng / mL TGF-β+50ng / mL IL-6 cytokines or 10ng / mL TGF-β+10ng / mL IL-2 cytokines were added to the T cell culture environment to induce CD4+T cells to differentiate into Th17 subsets and Treg subsets, respectively. At the same time, 10μg / mL ConPP was added to 1 / 3 of the well plate, and 10μg / mL SFRP2-PP96 was added to the other 1 / 3 of the well plate for co-treatment for 3 days. The cells were subjected to CD4 surface marker and IL-17A intracellular staining, and flow cytometry was used to detect Th17 subset staining; or the cells were subjected to CD4 and CD25 surface marker and Foxp3 nuclear transcription factor staining, and flow cytometry was used to detect Treg subset staining. After the machine detection, the lymphocyte gate, adhesion cell removal, and dead cell removal were performed in sequence using Flowjo10.4.0 software, and then CD4 was analyzed. + IL-17A + Th17 subset ratio and CD4 + CD25 + Foxp3 + The results showed that compared with the uninduced CD4 + T cells, after adding Th17 inducing cytokines, the proportion of Th17 subpopulation increased, and the proportion of Th17 subpopulation decreased slightly after SFRP2-PP96 treatment ( Figure 8 AB shown, Table 7); correspondingly, compared with uninduced CD4 + T cells, after the addition of Treg-inducing cytokines, the proportion of Treg subpopulation increased, and after SFRP2-PP96 treatment, the proportion of Treg subpopulation increased further (*P<0.05, Fig. 9 AB shown in Table 9). However, there was no significant change in the proportions of Th17 subsets and Treg subsets after ConPP treatment.

[0124] At the same time, total RNA was extracted from the remaining duplicate wells of the same batch of cells, and CD4 + Changes in the proliferation index Ki-67 and the relative expression levels of RORγt and Foxp3 mRNA during T cell induction and differentiation. The results showed that compared with uninduced CD4 + T cells, after adding Th17 inducing cytokines, the relative expression of RORγt mRNA increased significantly, and SFRP2-PP96 treatment inhibited the relative expression of RORγt mRNA (***P<0.005, Figure 8 C, Table 8); after adding Treg-inducing cytokines, the relative expression of Foxp3 mRNA increased significantly (***P < 0.005, Fig. 9C, Table 10). The relative mRNA expression of RORγt and Foxp3 after ConPP treatment did not change significantly.

[0125] Table 7

[0126]

[0127] Table 8

[0128]

[0129] Table 9

[0130]

[0131] Table 10

[0132]

[0133] Example 4 SFRP2-PP96 inhibits experimental colitis in mice

[0134] Twenty-four 6-8-week-old C57BL / 6J male mice were randomly divided into four groups: normal control group, 3%

[0135] DSS+PBS group, 3%DSS+ConPP group and DSS+SFRP2-PP96 group, 6 mice in each group. The weight, stool viscosity and blood in the stool of mice were recorded every day from the beginning of the administration of DSS drinking water. Compared with the mice in the normal control group, the DSS-induced experimental colitis mice showed obvious weight loss and dull fur from the 6th day, and obvious blood in the stool and diarrhea from the 4th day, which continued to worsen.

[0136] The weight change of mice on the day they started drinking DSS water (day 0) was regarded as 1, and the weight of mice was recorded every day. After the experiment, the ratio and decline rate of daily weight compared with day 0 were calculated, and a line graph of weight change of the four groups of mice was drawn ( Fig.10 A, Table 11). The line graph shows that: starting from the 6th day, the body weight of mice in the DSS+PBS group and the DSS+ConPP group decreased significantly until the end of the experiment (P < 0.01); the body weight change of mice in the DSS+SFRP2-PP96 group was not statistically different from that in the normal control group (P > 0.05), but was significantly higher than that in the DSS+PBS group and the DSS+ConPP group (P < 0.005). The results show that intraperitoneal injection of SFRP2 functional polypeptide can inhibit the weight loss trend of experimental colitis mice.

[0137] Taking the normal control group as the reference (regarded as 0), the disease activity index calculated for the four groups of mice was plotted as a line graph ( Fig.10B, Table 12). The line graph showed that starting from the 4th day, the DAI of mice in the DSS+PBS group and the DSS+ConPP group was significantly higher than that in the normal control group and the SFRP2 functional polypeptide treatment group (P < 0.005), while there was no significant difference in DAI between the SFRP2 functional polypeptide group and the normal control group (P > 0.05).

[0138] On the seventh day after the start of DSS drinking, the mice were killed and their colons, mesenteric lymph nodes, serum, feces and other tissues were collected. The colons of the four groups of mice were photographed from above ( Fig.10 As shown in Figure C, it can be seen that the colon of mice with experimental colitis induced by DSS was significantly shortened. The colon length of the ConPP treatment group was close to that of the DSS+PBS group. The colon of the SFRP2 functional polypeptide treatment group was longer than that of the DSS+PBS group, but still slightly shorter than that of the normal control group. The colon lengths of the four groups of mice were statistically analyzed and a bar graph was drawn ( Fig.10 D, Table 13), the results showed that the colon length of mice in the DSS+PBS group and the DSS+ConPP group was significantly lower than that in the normal control group (P < 0.01, P < 0.005) and the DSS+SFRP2-PP96 group (P < 0.01, P < 0.005), and there was no significant difference between the normal control group and the DSS+SFRP2-PP96 group (P > 0.05).

[0139] Then, the colon tissues of the four groups of mice were subjected to pathological H&E staining ( Fig.10 E), the results showed that the colon epithelial structure of the normal control group mice was intact, and there was no infiltration of inflammatory cells; compared with the normal control group, the DSS-induced experimental colitis group showed partial tissue mucosal or muscular layer defects, goblet cell loss, and infiltration of inflammatory cells such as lymphocytes and neutrophils; the DSS+ConPP group mice did not show a significant reduction in inflammatory infiltration; while the SFRP2-PP96 treatment group mice had a reduced degree of colon tissue mucosal damage, the mucosa or muscular layer was basically intact, the number of goblet cell losses was reduced, and the infiltration of inflammatory cells was significantly reduced.

[0140] The above H&E stained sections were scored for histological pathology ( Fig.10 As shown in F, Table 13), the colon pathological score of the mice in the DSS+PBS group was significantly higher than that in the normal control group (P<0.005), and the colon pathological score of the mice in the DSS+recombinant SFRP2 protein group was alleviated (P<0.01).

[0141] Table 11

[0142]

[0143]

[0144] Table 12

[0145]

[0146] Table 13

[0147]

[0148]

[0149] Example 5 SFRP2-PP96 inhibits Th17 proliferation and promotes Treg proliferation in colon tissue

[0150] The mesenteric lymph nodes of mice in each group were collected, and after adding cell stimulators (plus protein transport inhibitor, Invitrogen) to the cells for 5 hours, CD4 surface markers and IL-17A intracellular staining were performed; or the cells were directly stained for CD4 and CD25 surface markers and Foxp3 nuclear transcription factor staining. After flow cytometry detection, the lymphocyte gate, adhesion cells, and dead cells were removed in sequence using Flowjo 10.4.0 software, and the proportion of CD4+IL-17A+Th17 subpopulations and CD4+CD25+Foxp3+Treg subpopulations were analyzed. The results showed that compared with the normal control group, the proportion of Th17 subpopulations in CD4+T cells in the mesenteric lymph nodes of mice with DSS-induced colitis increased (P<0.01), and the proportion of Th17 subpopulations decreased after treatment with SFRP2 functional peptide SFRP2-PP96 (P<0.01), and the difference was statistically significant ( Fig.11 AB shown, Table 14). In addition, compared with the normal control group, the proportion of Treg subsets in CD4+T cells in the mesenteric lymph nodes of DSS-induced colitis mice was reduced (P < 0.05), and the proportion of Treg subsets was increased after treatment with SFRP2 functional peptide SFRP2-PP96 (P < 0.05), and the difference was statistically significant ( Fig.11 CD shown in Table 14). However, ConPP treatment had no significant effect on the proportion of Th17 subsets and Treg subsets (P>0.05).

[0151] In order to further explore the effect of SFRP2 functional polypeptide on the number of Th17 cells and Treg cells in colon tissue, immunohistochemical staining of RORγt and FOXP3 was performed on the paraffin sections of colon tissue of mice in each group. The results showed that the number of RORγt-positive cells in the colon tissue of the normal control group was relatively small, and the number of RORγt-positive cells in the colon tissue of the DSS+PBS group and the DSS+ConPP group was significantly increased (P < 0.005), while the number of RORγt-positive cells in the colon tissue of the DSS+SFRP2-PP96 treatment group was similar to that of the normal control group, and was significantly less than that of the DSS+PBS group and the DSS+ConPP group (P < 0.01, Fig.11 In addition, the number of FOXP3-positive cells in the colon tissue of the normal control group was relatively small, the number of FOXP3-positive cells in the colon tissue of the DSS+PBS group and the DSS+ConPP group was slightly increased, and the number of FOXP3-positive cells in the colon tissue of the DSS+SFRP2-PP96 group was significantly increased (P < 0.01, Fig.11 E, G shown, Table 15).

[0152] Table 14

[0153]

[0154] Table 15

[0155]

[0156] Example 6 SFRP2-PP96 inhibits the expression of pro-inflammatory proteins and promotes the expression of anti-inflammatory proteins in inflamed colon tissue

[0157] A section of colon tissue was collected from each group of mice for inflammation-related protein chip detection. A total of 18 protein expression levels were detected.

[0158] Further quantitative analysis results showed that there were significant differences in the expression of four proteins in the colon tissues of each group, namely IL-17A, IL-22, IL-10 and TGF-β1. The bar graph results showed that the expression levels of pro-inflammatory proteins IL-17A and IL-22 in the colon tissues of colitis mice were significantly higher than those in the normal control group (P < 0.05, Fig.12 A and B shown in Table 16), after ConPP treatment, the expression levels of IL-17A and IL-22 did not decrease significantly (P>0.05, Fig.12 A and B shown in Table 16), and their expression levels were significantly decreased after SFRP2-PP96 treatment (P < 0.05, Fig.12In addition, the expression levels of anti-inflammatory proteins IL-10 and TGF-β1 in the colon tissues of colitis mice were significantly lower than those in the normal control group (P < 0.05, P < 0.005, Fig.12 C and D shown in Table 16), the control peptide ConPP treatment had no significant effect on its expression level (P>0.05, Fig.12 C and D shown in Table 16), while after treatment with SFRP2 functional polypeptide SFRP2-PP96, the expression levels of IL-10 and TGF-β1 were significantly increased (P < 0.05, P < 0.01, Fig.12 C, D shown, Table 16).

[0159] Table 16

[0160]

[0161] Example 7 SFRP2-PP96 may promote recovery from colitis by regulating the proportion of intestinal flora

[0162] The intestinal contents of each group of mice were sequenced and analyzed by 16S rDNA and the data were integrated. We also analyzed the fecal microorganisms of each group of mice at the phylum and genus levels. Fig.13 As shown in A, in the DSS+PBS group and the DSS+ConPP group, compared with the normal control group, the number of Firmicutes (orange) and Bacteroidetes (blue) decreased, and the ratio of Firmicutes to Bacteroidetes also decreased (p < 0.05, Fig.13 B, Table 17); In the DSS+SFRP2-PP96 group, compared with the normal control group, the number of Firmicutes (orange) and Bacteroidetes (blue) decreased, but the ratio of Firmicutes / Bacteroidetes increased to a level close to that of the normal control group, and was significantly higher than that of the DSS+PBS group and the DSS+ConPP group (P < 0.01, P < 0.05, Fig.13 B, Table 17), indicating that SFRP2 functional peptide treatment can restore the balance of some intestinal flora.

[0163] At the genus level, the main bacteria in the normal control group were Muribaculaceae, and the relative abundance of Muribaculaceae in the DSS+PBS group was lower than that in the normal control group (P<0.005, Fig.13 C and D, Table 17), the relative abundance of Muribaculaceae did not change significantly after ConPP treatment, but after SFRP2-PP96 treatment, the relative abundance of Muribaculaceae increased significantly (P < 0.01, Fig.13C and D shown in Table 17). The changing trend of Lactobacillus (Lachnospiraceae) in the four groups of mice was similar to that of Muribaculaceae (P < 0.01, Fig.13 In the DSS+SFRP2-PP96 group, Dubosiella became the dominant bacteria, which was significantly higher than that in the other two groups (P<0.05, P<0.005, Fig.13 C, F shown, Table 17).

[0164] The above results prove that SFRP2-PP96 helps to improve intestinal flora imbalance and promote the recovery of enteritis.

[0165] Table 17

[0166]

[0167] Example 8 SFRP2-PP96 treats experimental periodontitis in mice

[0168] After 3 weeks of SFRP2 functional peptide injection, the mice were killed and the maxillae on both sides were removed. The soft tissue of the palate was cut to extract RNA, and the maxillary tissue was fixed with 4% paraformaldehyde and then micro-CT was taken. The therapeutic effect on experimental periodontitis in mice was evaluated by imaging.

[0169] The 3D stereo imaging results are as follows Fig.14 , the height from the cementoenamel junction to the alveolar ridge top was measured, i.e., clinical attachment loss (AL, CEJ-AB). The results are shown in Table 18. Compared with the normal control group, the CEJ-AB of the mice in the NaCl control group and the ConPP treatment group increased significantly (P < 0.01), while the CEJ-AB of the SFRP2-PP96 treatment group was restored (P < 0.05).

[0170] Table 18

[0171]

[0172]

[0173] Example 9 SFRP2-PP96 promotes odontogenic differentiation of DPSCs in vitro and can partially rescue the odontogenic differentiation of SFRP2sh DPSCs

[0174] In order to study the effect of SFRP2 functional polypeptide on the odontogenic differentiation of DPSCs in vitro, alkaline phosphatase (ALP) activity assay, Alizarin red staining, calcium ion quantification and Western blot experiments were performed.

[0175] 15% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 2 mmol / L L-glutamine, 0.05 mmol / L ascorbic acid, 10 mmol / L sodium β-glycerophosphate, and 10 nmol / L dexamethasone were added to the α-MEM culture medium to prepare the tooth induction medium. 10 μg / mL ConPP (control polypeptide) and SFRP2-PP96 were added to the culture medium. DPSCs were taken at 1×10 5 The cells were inoculated into 6-well plates at a concentration of 100 cells / well. When the cells grew to 70%, the tooth induction medium was replaced. The medium was changed every 3 days.

[0176] At 7 days after odontogenesis, the alkaline phosphatase activity of DPSCs was determined using an ALP activity kit (Sigma-Aldrich, USA). The optical density (OD) value was measured using a microplate reader (absorbance 405 nm), and the ALP activity of the sample was calculated using the standard ALP activity curve. The results showed that ConPP had no significant effect on the ALP activity of DPSCs (P>0.05, Fig.17 A), while SFRP2-96 peptide significantly enhanced the ALP activity of DPSCs (P<0.01, Fig.15 A).

[0177] After 2 weeks of odontogenesis induction culture, the cells were fixed with tissue fixative (Biyuntian) for 1 hour and stained with 2% alizarin red dye (Sigma-Aldrich, USA). The calcium ion concentration was also measured: 10% cetylpyridinium chloride (dissolved in 10mmol / L sodium phosphate) was used at 20℃±5℃ for 30 minutes to decolorize the alizarin red dye, and the OD value was measured using an enzyme reader (absorbance 562nm), and the calcium ion concentration of the sample was calculated using the standard calcium ion concentration curve. The results showed that compared with the control group, ConPP had no significant effect on the alizarin red staining intensity and calcium ion concentration of DPSCs (P>0.05, Fig.15 B- Fig.15 C), while SFRP2-96 peptide significantly enhanced the Alizarin red staining intensity and calcium ion concentration of DPSCs (P<0.01, Fig.15 B Fig.15 -C).

[0178] At 7 days after odontogenesis induction, intracellular proteins were extracted and the intracellular DSPP and DMP1 protein contents were detected by Western blot. The results showed that ConPP had no significant effect on the intracellular DSPP and DMP1 protein contents of DPSCs ( Fig.15 D), while SFRP2-96 peptide significantly increased the intracellular DSPP and DMP1 protein content in DPSCs ( Fig.15 D).

[0179] To study the rescue effect of SFRP2 functional peptide on SFRP2 knockdown DPSCs, alkaline phosphatase (ALP) activity assay, Alizarin red staining and Western blot experiments were performed.

[0180] Short hairpin RNAs (shRNAs) complementary to the sequence of the target gene SFRP2 were subcloned into the pLKO.1 lentiviral vector (Addgene, USA), and Scramble shRNAs (Scramsh) were used as knockout control viruses. The target sequence of SFRP2shRNAs is as follows: 5'-ttgatgtaggttatctccttc-3'. Scramble shRNAs and SFRP2shRNAs were transfected into DPSCs, and the SFRP2 knockdown efficiency was detected by Real-time RT-PCR. The results showed that the expression of SFRP2 RNA in the SFRP2sh group was significantly reduced (P<0.01, Fig.16 A). Stably transfected DPSCs were induced into odontogenesis, and 10 μg / mL SFRP2-PP96 was added to the SFRP2sh group. ALP activity was detected at 5 days after odontogenesis, and it was found that the ALP activity in the SFRP2sh group was significantly reduced compared with that in the Scramsh group (P<0.01, Fig.16 B), but it could be partially rescued by SFRP2-PP96 (P<0.05, Fig.16 B). Similarly, the results of alizarin red staining at 2 weeks after odontogenesis induction showed that the depth of alizarin red staining in the SFRP2sh group was significantly reduced compared with the Scramsh group ( Fig.16 C), but can be partially rescued by SFRP2-PP96 ( Fig.16 C). In addition, the results of protein expression at 7 days after odontogenesis induction showed that the expression of intracellular DSPP and DMP1 proteins decreased after SFRP2 knockout ( Fig.16 D), but can be partially rescued by SFRP2-PP96 ( Fig.16 D).

[0181] Example 10 SFRP2-PP96 promotes DPSCs-mediated tooth regeneration in the jaw

[0182] To study the effect of SFRP2-PP96 on DPSCs-mediated tooth regeneration in the jaw, the right maxillary and mandibular incisors of rabbits were extracted, and a mixture of 10ug / ml ConPP or SFRP2-PP96 cells and hydrogel scaffolds was reimplanted and sutured to establish a rabbit tooth regeneration model in the jaw ( Fig.17 ).

[0183] The mice were killed 3 months later, and microCT was taken to calculate the volume of high-density calcifications in the jawbone. The results showed that ConPP had no significant effect on the volume of high-density calcifications in the jawbone compared with the control group (P>0.05, Fig.18 A. Fig.18 B), while the volume of calcified regeneration density in the jaws of the SFRP2-PP96-treated group increased significantly (P<0.05, Fig.18 A. Fig.18 B). The results of gross observation of the jaw section showed that there was no significant difference between the ConPP group and the control group, while the high-density calcification formed in the SFRP2-96 peptide treatment group was significantly more than that in the control group ( Fig.18 C).

[0184] Further histological staining of rabbit jaw sections showed that there was no significant difference between the ConPP group and the control group, while the SFRP2-96 peptide-treated group formed a large number of dentin-like regenerations surrounded by a large number of odontoblast-like cells ( Fig.18 D). The results of Masson staining also showed that there was no significant difference between the ConPP group and the control group, but the treatment with SFRP2-96 peptide formed more collagen and osteoid ( Fig.18 E). In addition, the results of immunohistochemistry and immunofluorescence staining showed that the expression intensity of DSPP and DMP1 in the jawbone of the SFRP2-96 peptide treatment group was stronger ( Fig.18 F. Fig.18 G), there was no significant difference between the ConPP group and the control group ( Fig.18 F. Fig.18 G).

[0185] Example 11 SFRP2-PP96 inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a

[0186] To explore the binding ability of SFRP2-PP96 to Wnt3a, microscale thermophoresis (MST) experiments were used for verification. Wnt3a recombinant protein and SFRP2-PP96 were placed in a capillary according to a certain concentration gradient. Infrared laser heating produced a microscopic temperature gradient field for thermophoresis, causing changes in the fluorescence distribution in the reaction system. The results showed that the Kd value was 8.723E-6, and SFRP2-PP96 could bind to Wnt3a in vitro ( Fig.19 A).

[0187] The effect of SFRP2-PP96 on intracellular Wnt / β-catenin in DPSCs was further explored. ConPP and SFRP2-PP96 were added to the culture medium, and intracellular proteins were collected after 24 hours of treatment, and Western blot experiments were performed for verification. The results showed that the total intracellular β-catenin was significantly different among the three groups, but SFRP2-PP96 enhanced the intracellular p-β-catenin level and inhibited the Wnt / β-catenin signaling pathway ( Fig.19 B), ConPP had no significant effect on intracellular p-β-catenin levels ( Fig.19 B). In addition, the level of intracellular p-β-catenin decreased after SFRP2 knockout, but increased after the addition of SFRP2-PP96 ( Fig.19 C).

[0188] Therefore, it was concluded that SFRP2-PP96 inhibited the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a.

[0189] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. SFRP2 polypeptide, characterized in that have: (I), the amino acid sequence shown in SEQ ID No. 1 or 2; or (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (I).

2. Use of the SFRP2 polypeptide according to claim 1 in any of the following: (1) regulating the osteogenic and / or odontogenic differentiation of mesenchymal stem cells; and / or (2) Promote tissue repair and regeneration and / or regulate immune balance.

3. Use of the SFRP2 polypeptide according to claim 1 in the preparation of a medicament for preventing and / or treating periodontitis.

4. The use according to claim 3, characterized in that The prevention and / or treatment of periodontitis comprises reducing CD4 + Increased proportion of Th17 subsets and / or CD4 + The proportion of Treg subsets in T cells; The method for preventing and / or treating periodontitis further comprises inhibiting CD4 + T cells differentiate into Th17 subsets and / or promote CD4 + T cells differentiate into Treg subsets.

5. Use of the SFRP2 polypeptide according to claim 1 in the preparation of a medicament for preventing and / or treating colitis.

6. The use according to claim 5, characterized in that The prevention and / or treatment of colitis comprises inhibiting CD4 + Proliferation of Th17 subsets in T cells and / or promotion of CD4 + Proliferation of Treg subset in T cells.

7. The use according to claim 5 or 6, characterized in that The prevention and / or treatment of colitis includes inhibiting the expression of pro-inflammatory proteins and / or promoting the expression of anti-inflammatory proteins.

8. The use according to any one of claims 5 to 7, characterized in that The prevention and / or treatment of colitis includes regulating the ratio of intestinal flora.

9. A drug, characterized in that Comprising the SFRP2 polypeptide as claimed in claim 1.

10. A pharmaceutical combination, characterized in that The method comprises the drug as claimed in claim 9 and any other active ingredients.

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