SFRP2 functional polypeptide and use thereof

Abstract

The present invention relates to an SFRP2 functional polypeptide and a use thereof. Provided are an SFRP2 functional polypeptide and a use thereof. The SFRP2 functional polypeptide (SFRP2-PP96) can reduce the proportion of a Th17 subset in CD4+ T cells and increase the proportion of a Treg subset, inhibit the differentiation of induced CD4+ T cells into the Th17 subset and promote the differentiation into the Treg subset, inhibit experimental colitis in mice, inhibit the proliferation of Th17 in colon tissues and promote the proliferation of Treg, inhibit the expression of a pro-inflammatory protein in an inflammatory colon tissue and promote the expression of an anti-inflammatory protein, promote the recovery of colitis by adjusting the proportion of intestinal flora, and enhance the therapeutic effect of experimental periodontitis. In addition, the SFRP2 functional polypeptide is a polypeptide preparation, has superior characteristics to traditional full-length proteins, and is convenient for subsequent clinical application and popularization.

Description

SFRP2 functional polypeptide and its application

[0001] This application claims priority to 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, typically presenting in early adulthood. Persistent inflammation of the periodontium can lead to deepening of periodontal pockets, periodontal suppuration, bone loss, and tooth loss. Repair of periodontal bone defects remains a major challenge in the field of regenerative medicine. Mild to moderate periodontitis is generally managed with nonsurgical therapies and oral hygiene education. Severe periodontitis requires surgical treatment, but traditional treatments such as guided bone regeneration and guided tissue regeneration have limited and inconsistent regenerative effects, failing to achieve complete regeneration of the periodontium. Furthermore, in periodontitis, a complex immune response, encompassing both innate and adaptive immune responses, is activated in the periodontium to control pathogen invasion. However, dysregulation of both the innate and adaptive immune systems may play a significant role in the etiology of periodontitis. Cytokines produced by the immune response regulate the interactions and cellular networks between macrophages, T cells, B cells, epithelial cells, and other cell types. Proinflammatory cytokines activate immune defenses 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 treatments, and immunotherapy can serve as an adjunct or alternative to traditional treatments. Immunotherapy has been shown to potentially regulate dysregulated immunity in autoimmune and inflammatory diseases. Current treatments for periodontitis focus primarily on periodontal cleaning and scaling, or sometimes require combined antibiotic treatment to remove biofilms. However, these therapies often fail to completely eliminate periodontal pathogens, and their continued presence continues to stimulate the host's immune response. In turn, the host's dysregulated immune response leads to the 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, through STAT6, induces Treg differentiation and downregulates Th17 differentiation. The dynamic balance between Th17 and Treg is a crucial regulatory factor in inflammation. To date, the causes of Th17 / Treg imbalance during periodontitis remain unclear.

[0005] The Wnt / β-catenin signaling pathway plays a key role in tooth development and differentiation. However, the role of the Wnt / β-catenin signaling pathway in periodontitis remains controversial.

[0006] Secreted frizzled-related protein 2 (SFRP2), as an antagonist of the Wnt / β-catenin signaling pathway, has many advantages, including 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 cycles, and high production costs. In addition, they cannot introduce non-natural amino acids and cannot be amidated at their termini, which limits their further clinical translation 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 polypeptides and their applications. SFRP2 functional polypeptides (SFRP2-PP96) can reduce the proportion of Th17 subpopulations in CD4+ T cells and increase the proportion of Treg subpopulations; inhibit the differentiation of induced CD4+ T cells into Th17 subpopulations and promote differentiation into Treg subpopulations; inhibit experimental colitis in mice; inhibit Th17 proliferation in colonic tissue and promote Treg proliferation; inhibit the expression of pro-inflammatory proteins in inflamed colonic tissue and promote the expression of anti-inflammatory proteins; promote colitis recovery by regulating the proportion of intestinal flora; and enhance the therapeutic effect of experimental periodontitis. Furthermore, as a polypeptide preparation, it has properties superior to traditional full-length proteins, facilitating 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 an 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 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 T cells + 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 the odontogenic differentiation ability of SFRP2sh DPSCs;

[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 subsets 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 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 and / or Dunaliella.

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

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

[0042] The present invention also provides a method for preventing and / or treating periodontitis, comprising administering any one of the following:

[0043] (I), the SFRP2 polypeptide;

[0044] (II), the drug; or

[0045] (III), the drug combination.

[0046] The present invention also provides a method for preventing and / or treating colitis, comprising administering any one of the following:

[0047] (I), the SFRP2 polypeptide;

[0048] (II), the drug; or

[0049] (III), the drug combination.

[0050] The present invention provides a functional SFRP2 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

[0051] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0052] Figure 1 shows a histogram of the grayscale values ​​of the peptide chip in Effect Example 1;

[0053] Figure 2 shows the peptide chip array results in Effect Example 1;

[0054] Figure 3 shows the purity, molecular weight, and solubility information of the control peptide ConPP and the functional peptide SFRP2-PP96 in Example 1;

[0055] FIG4 shows the in vitro osteogenic induction results of different polypeptide groups in the experimental example;

[0056] FIG5 shows the results of the in vitro migration ability of human dental pulp stem cells in the experimental example of SFRP2 functional polypeptide;

[0057] FIG6 shows the results of the in vitro chemotactic ability of SFRP2 functional polypeptide on human dental pulp stem cells in the experimental example;

[0058] FIG7 shows the detection results of SFRP2 functional polypeptide on Th17 and Treg subsets in Example 2;

[0059] FIG8 shows the detection results of SFRP2 functional polypeptide on Th17 and Treg subsets under Th17 polarization conditions in Example 3;

[0060] FIG9 shows the detection results of SFRP2 functional polypeptide on Th17 and Treg subsets under Treg polarization conditions in Example 3;

[0061] FIG10 shows the results of the treatment of experimental colitis in mice by the SFRP2 functional polypeptide in Example 4;

[0062] FIG11 shows the detection results of SFRP2 functional polypeptide on Th17 and Treg subsets in mesenteric lymph nodes and colon tissues in Example 5;

[0063] FIG12 shows the detection of inflammation-related proteins in colon tissue by SFRP2 functional polypeptide in Example 6;

[0064] FIG13 shows the effect of SFRP2 functional polypeptide on regulating intestinal flora in Example 7;

[0065] FIG14 shows 3D stereoscopic imaging of the therapeutic effect of SFRP2 functional polypeptide on experimental periodontitis in mice in Example 8;

[0066] FIG15 shows the results of the in vitro odontogenic differentiation of human dental pulp stem cells by the SFRP2 functional polypeptide in Example 9;

[0067] FIG16 shows that the SFRP2 functional polypeptide in Example 9 partially rescues the odontogenic differentiation ability of SFRP2sh dental pulp stem cells in vitro;

[0068] FIG17 shows the method for establishing the rabbit jaw tooth regeneration model in Example 10;

[0069] FIG18 shows the ability of the SFRP2 functional polypeptide in Example 10 to promote tooth regeneration in rabbit jaws mediated by dental pulp stem cells;

[0070] FIG19 shows that the SFRP2 functional polypeptide in Example 11 inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a. DETAILED DESCRIPTION

[0071] The present invention discloses SFRP2 functional polypeptides and their applications. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that those skilled in the art can 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.

[0072] The present invention discloses SFRP2 functional polypeptides and their applications. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that those skilled in the art can 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.

[0073] The chemical synthesis technology for peptides is now increasingly mature, with low R&D and synthesis costs. Peptide products are easily separated from impurities, resulting in high purity. Furthermore, peptides can be easily modified by introducing unnatural amino acids during the production process. Because peptides are primarily degraded into amino acids through proteolysis and eliminated by renal filtration, their metabolites are non-toxic.

[0074] 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.

[0075] After initially clarifying the regulatory role of the full-length SFRP2 protein in Th17 / Treg balance and its therapeutic effects on periodontitis, we discovered through website searches and literature searches that the Fz region of the SFRP2 protein can bind to Wnt ligands. Identifying the binding fragments of SFRP2 for Wnt ligands for more precise follow-up research and clinical translation remains an urgent issue. Using peptide microarray analysis, we report for the first time the key interaction sites for SFRP2 binding to Wnt ligands. Peptide microarray results revealed two sites with strong binding: sites 7-8 (designated SFRP2-PP95, amino acid sequence: LQLCHGIEYQNMRLPNL, SEQ ID No. 1) and sites 49-54 (designated SFRP2-PP96, amino acid sequence: AFGFPWPDMLECDRFPQDNDLCIPL, SEQ ID No. 2). We subsequently synthesized these two peptides and used ConPP (amino acid sequence: EEEACDQQPQEEEEKDEEGE, SEQ ID No. 3) for comparison. SFRP2 functional peptides can block the Wnt signaling pathway by binding to the putative Wnt ligand of the Frizzled receptor. To screen for the optimal concentration of functional peptides acting on cells, based on the results of an in vitro osteogenic differentiation experiment on human dental pulp stem cells (DPSCs), the peptide targeting sites 49-54 (i.e., SFRP2-PP96) was selected for subsequent experimental studies.

[0076] 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.

[0077] 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.

[0078] The present invention will be further described below in conjunction with the embodiments:

[0079] Preparation Example Peptide Array Chip Synthesis:

[0080] 1. Peptide array chip synthesis:

[0081] A peptide array was synthesized based on the sequence of SFRP2 protein (SEQ ID NO: Q96HF1). Two peptide array chips were prepared according to the overlapping design and peptide array chip SOP (Standard Operating Procedure):

[0082] (1) The activated matrix chip membrane was placed on a fully automatic peptide chip synthesizer (Aurora Group Company, VERSA 110). According to the program, Fmoc-amino acid solution (Chengdu Chengnuo Biotechnology Co., Ltd., 20 types) was automatically transferred to a specific position on the activated membrane to react with the membrane;

[0083] (2) After each layer was synthesized, the membrane was immersed in blocking solution I (a solution of DMF (N,N-Dimethylformamide, anhydrous, amine free, 99.9%, Alfa-Asia Product No.: 043465) containing 2% (v / v) acetic anhydride (Sinopharm Chemical Reagent Co., Ltd., Product No.: 10000317)) and blocking solution II (a solution of DMF containing 2% (v / v) acetic anhydride and 2% (v / v) DIPEA (N,N-Diisopropylethylamine, Aldrich Product No.: 550043)) for side chain blocking. The membrane was then washed with DMF (N,N-dimethylformamide, Sinopharm Chemical Reagent Co., Ltd., Product No.: CST12767S) five times for 3 minutes each time.

[0084] (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 terminus. After deprotection, the membrane was washed with DMF five times for 3 minutes each, then washed with ethanol (Sinopharm Chemical Reagent Co., Ltd., Catalog No.: 100092008) five times for 3 minutes each, and dried. The above steps were repeated until the peptide array was fully synthesized.

[0085] (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 for 3 minutes each time. Then, the membrane is washed with CH2Cl2 (dichloromethane, Sinopharm Chemical Reagent Co., Ltd., catalog number: 8004731916) 5 times for 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 for 3 minutes each time, and finally washed with ethanol, washed with DMF 5 times for 3 minutes each time, and dried naturally. The completed peptide array chip is used immediately or stored at -20℃ / -80℃.

[0086] Example 1 Polypeptide Array Chip and Serum Immune Response Detection

[0087] (1) Peptide array chip sealing:

[0088] After activating the peptide array chip, add blocking solution and shake at 20℃±5℃ for 4 hours;

[0089] (2) Incubation of the protein sample with the peptide array chip: The tested 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, while the control group used blocking solution. Incubate at 4°C overnight with shaking. Remove the membrane and wash the membrane five times with TBST (Tris Buffered Saline with Tween 20) for 10 minutes each time.

[0090] (3) Incubation of peptide array chip with primary antibody: After the primary antibody (Anti-Wnt3a antibody) was diluted 1:1000, 10 mL of each of the experimental group and the control group were incubated at 20℃±5℃ for 2 hours, and the membrane was washed 5 times, each time for 10 minutes;

[0091] (4) Incubation of peptide array chip with HRP-labeled secondary antibody: After HRP-labeled secondary antibody was diluted 1:10000, the experimental group and the control group were incubated with 10 mL each, shaken at 20℃±5℃ for 2 hours, and the membrane was washed 5 times, each time for 10 minutes;

[0092] (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 (VILBER FUSION FX7 Spectra, Vilber, France).

[0093] Effect Example 1

[0094] 1. Synthesis of peptides

[0095] According to the array results, the sequences corresponding to the obvious color spots were selected for peptide synthesis and subsequent functional verification.

[0096] 2. The array results are shown in Table 1, Figures 1 and 2.

[0097] Table 1

[0098] From the data in Figures 1 and 2, it can be analyzed that points 7-8 (amino acid sequence: LQLCHGIEYQNMRLPNL, SEQ ID No. 1) and points 49-54 (amino acid sequence: AFGFPWPDMLECDRFPQDNDLCIPL, SEQ ID No. 2) on the polypeptide array have color development effects.

[0099] Subsequently, a peptide concentration of 10 μg / mL was used for in vivo and in vitro experimental studies.

[0100] 3. The purity, molecular weight, and solubility of the control polypeptide ConPP and the functional polypeptide SFRP2-PP96 are shown in Figure 3. Both the control polypeptide ConPP and SFRP2-PP96 are polypeptides produced in the present invention.

[0101] 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.

[0102] 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.

[0103] Experimental Example: Selection of SFRP2-PP95 and SFRP2-PP96

[0104] Fourth-generation (P4) DPSCs were used for in vitro osteogenic induction: trypsin (Gibco) digestion and centrifugation were performed, and the cells were resuspended in culture medium containing fetal bovine serum (FBS, Gibco) and counted on a hemocytometer to a cell density of 1 × 10 cells / well in a 6-well plate. 5 / well. When the cells grew to a density of 70%, the culture medium was aspirated, and the cells were washed with phosphate buffered saline (PBS, Biosharp) and replaced with osteogenic induction medium. The osteogenic induction medium was replaced every 2 to 3 days. Among them, the DPSCs group served as the control group, the DPSCs+ConPP group maintained 10 μL / mL ConPP in the culture medium during the osteogenic induction process, the DPSCs+SFRP2-PP95 group maintained 10 μL / mL SFRP2-PP95 in the culture medium during the osteogenic induction process, and the DPSCs+SFRP2-PP96 group maintained 10 μL / mL SFRP2-PP96 in the culture medium during the osteogenic induction process. 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 was aspirated and the cells were rinsed three times with PBS. The appearance of mineralized nodules was observed under an inverted microscope and pictures were collected.

[0105] A 10% aqueous solution of CPC (cetylpyridinium chloride, homemade) was added to the plate to dissolve Ca2+ ions, 1 mL per well, and the cells were incubated at 20°C ± 5°C for 30 minutes. The solution was diluted 1:10, and the absorbance (OD) was measured at a wavelength of 562 nm in a microplate reader. Data were analyzed using SPSS 22.0 and Prism 7.0 software. One-way ANOVA was used for statistical analysis, and data are expressed as mean ± SEM. Statistical significance was considered when P < 0.05.

[0106] Fourth-generation (P4) DPSCs were used for in vitro osteogenic induction: trypsin (Gibco) digestion and centrifugation were performed, and the cells were resuspended in culture medium containing fetal bovine serum (FBS, Gibco) and counted on a hemocytometer to a cell density of 1 × 10 cells / well in a 6-well plate. 5 / well. When the cells grow to a density of 70%, the culture medium is aspirated, 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. The grouping is the same as above. After 7 consecutive days of induction, the culture medium is discarded, the cells are washed three times with PBS, 600μL lysis buffer (Biyuntian) is added, and the cells are incubated at 37°C for 15 minutes. The cells are scraped and transferred into a 1.5mL EP tube, and centrifuged at 14000rpm and 4°C for 10 minutes. 50μL Alkaline buffer sol and 50μL Stock Substrate sol are added to each well of the 96-well plate, 10μL of the lysed cell sample is added and mixed, and the mixture is incubated at 37°C for 15 minutes. The absorbance of the well plate is detected at 405nm.

[0107] The results showed that the DPSCs+SFRP2-PP95 and DFSCs+SFRP2-PP96 groups exhibited enhanced mineralization capacity compared to the control group and the control peptide group (Figure 4A). Calcium ion quantification results showed that 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, with the effect of SFRP2-PP96 being more significant than that of SFRP2-PP95 (Figure 4B, Table 2). ALP activity results showed that the SFRP2-PP96 group exhibited higher ALP activity, while the SFRP2-PP95 group did not show statistically significant differences compared to the control group and the control peptide group (Figure 4C, Table 2).

[0108] Table 2

[0109] Cell scratch test: DPSCs were cultured at a rate of 5 × 10 4 Cells were seeded at a density of 100 μg / well in a 6-well plate and allowed to grow to 90% confluence. After 24 hours of serum-free culture, a cross-scratch was made along the well diameter using a 10 μL pipette tip, followed by the addition of fresh culture medium. The scratches were observed microscopically at the same viewing angle at 0, 24, and 48 hours to assess the degree of wound healing. ImageJ 1.49v software was used to detect blank areas, and the degree of wound healing was determined based on the percentage of blank areas in each group.

[0110] Cell Transwell experiment: Prepare a 0.1% collagen solution at a concentration of 6-10 μg / cm 2 Coat the Transwell chamber with a concentration of 100 μg / mL and incubate at 37°C for several hours. Remove excess liquid from the coated surface and allow to dry overnight. Add serum-free culture medium containing BSA and incubate at 37°C for 30 minutes. Then aspirate the remaining liquid in the culture plate. 5 100 μL of a DPSC cell suspension (100 μL / mL) was added to the upper chamber of a Transwell plate. DPSCs were cultured in a serum-free environment for 24 hours before preparation of the cell suspension. 600 μL of α-MEM medium supplemented with 5% fetal bovine serum was added to the lower chamber of a 24-well plate. Incubate for 6 hours as usual. Remove the Transwell chamber, discard the culture medium, wash twice with calcium-free PBS, fix with methanol for 30 minutes, and air-dry the chamber. Stain with 0.1% crystal violet for 20 minutes. Gently remove unmigrated cells from the upper layer with a cotton swab, and wash three times with PBS. Observe five fields of view under a 400x microscope and count the cells.

[0111] The results showed that at 24, 48, and 72 hours, the DPSCs + SFRP2-PP95 group exhibited stronger migration ability than the control group and the control peptide group (Figure 5A, B, C, D, Table 3); at 24 and 72 hours, the DPSCs + SFRP2-PP96 group exhibited stronger migration ability than the control group and the control peptide group (Figure 5A, B, D). In addition, at 24 and 48 hours, the DPSCs + SFRP2-PP95 group exhibited stronger chemotactic ability than the control group and the control peptide group (Figure 6A, B, C, Table 4); at 24 hours, the DPSCs + SFRP2-PP96 group exhibited stronger chemotactic ability than the control group and the control peptide group (Figure 6A, B).

[0112] Table 3

[0113] Table 4

[0114] The above results showed that SFRP2-PP96 peptide may have stronger biological functions, such as promoting the osteogenic differentiation of DPSCs. Therefore, SFRP2-PP96 was selected as the SFRP2 functional peptide in subsequent experiments.

[0115] Example 2 SFRP2-PP96 reduces the proportion of Th17 subsets and increases the proportion of Treg subsets in CD4+ T cells

[0116] In order to study the effects of SFRP2 functional polypeptide on CD4+T cell subsets, especially Th17 and Treg subsets, flow cytometry and Real-time RT-PCR were used for detection.

[0117] First, CD4 + T cells, in CD4 + T cells were cultured in a 10 μg / mL ConPP (control peptide) and SFRP2-PP96 environment for 3 days, and the cells were stained for CD4 surface markers and IL-17A intracellularly, and the Th17 subpopulation staining was detected by flow cytometry; or the cells were stained for CD4 and CD25 surface markers and Foxp3 nuclear transcription factor staining, and the Treg subpopulation staining was detected by flow cytometry. After the test, the lymphocyte gate, adherent cells, and dead cells were removed in sequence using Flowjo 10.4.0 software, and the CD4 + IL-17A + Th17 subset ratio and CD4 + CD25 + Foxp3 +The results showed that compared with the blank control group, the CD4 + The proportions of Th17 and Treg subpopulations in T cells did not change significantly (P>0.05, AB and DE in Figure 7), but the CD4 + The proportion of Th17 subpopulation in T cells decreased (P < 0.05, AB shown in Figure 7, Table 5), and the proportion of Treg subpopulation increased (P < 0.05, DE shown in Figure 7, Table 5).

[0118] At the same time, total RNA was extracted from the remaining 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. The results showed that compared with the blank control group, CD4 + The relative mRNA expression levels of RORγt and Foxp3 in T cells did not change significantly (P>0.05, Figure 7C, F), but the relative mRNA expression levels of RORγt and Foxp3 in CD4 T cells treated with SFRP2-PP96 did not change significantly (P>0.05, Figure 7C, F). + The relative expression level of RORγt mRNA in T cells was significantly decreased (P < 0.01, Figure 7C, Table 6), and the relative expression level of Foxp3 mRNA was significantly increased (P < 0.01, Figure 7F, Table 6).

[0119] 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 peptide had no such effect.

[0120] Table 5

[0121] Table 6

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

[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 well plate, and 10μg / mL SFRP2-PP96 was added to the other 1 / 3 well plate for co-treatment for 3 days. The cells were stained for CD4 surface markers and IL-17A intracellularly, and the Th17 subset staining was detected by flow cytometry; or the cells were stained for CD4 and CD25 surface markers and Foxp3 nuclear transcription factor staining, and the Treg subset staining was detected by flow cytometry. After the machine detection, the lymphocyte gate, adhesion cells, and dead cells were removed in sequence using Flowjo 10.4.0 software, and the CD4 + 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 subsets increased, and the proportion of Th17 subsets after SFRP2-PP96 treatment was slightly reduced (Figure 8 AB, Table 7); correspondingly, compared with uninduced CD4 + In T cells, the proportion of Treg subsets increased after the addition of Treg-inducing cytokines, and further increased after SFRP2-PP96 treatment (*P < 0.05, AB in Figure 9, Table 9). However, the proportions of Th17 and Treg subsets did not change significantly after ConPP treatment.

[0124] At the same time, total RNA was extracted from the remaining wells of the same batch of cells and CD4 + The changes of Ki-67, a proliferation index, and the relative expression levels of RORγt and Foxp3 mRNA during T cell differentiation were analyzed. + In T cells, the relative mRNA expression of RORγt was significantly increased after the addition of Th17-inducing cytokines, and SFRP2-PP96 treatment inhibited the relative mRNA expression of RORγt (***P < 0.005, Figure 8C, Table 8). In contrast, the relative mRNA expression of Foxp3 was significantly increased after the addition of Treg-inducing cytokines (***P < 0.005, Figure 9C, Table 10). The relative mRNA expression of RORγt and Foxp3 did not change significantly after ConPP treatment.

[0125] Table 7

[0126] Table 8

[0127] Table 9

[0128] Table 10

[0129] Example 4 SFRP2-PP96 inhibits experimental colitis in mice

[0130] Twenty-four 6- to 8-week-old C57BL / 6J male mice were randomly divided into four groups (n=6): a normal control group, a 3% DSS + PBS group, a 3% DSS + ConPP group, and a DSS + SFRP2-PP96 group. Starting with the administration of DSS in the drinking water, the mice's body weight, stool consistency, and blood in the stool were recorded daily. Compared with the normal control group, mice with DSS-induced experimental colitis showed significant weight loss and dull fur from day 6. Furthermore, they developed significant blood in the stool and diarrhea from day 4, which progressively worsened.

[0131] The weight change on the day mice began drinking DSS (day 0) was considered 1. Daily weights were recorded, and the ratio and rate of weight loss compared to day 0 were calculated after the end of the experiment. Line graphs of weight changes for the four groups of mice were plotted (Figure 10A, Table 11). The line graphs showed that starting on day 6, the weight of mice in the DSS+PBS and DSS+ConPP groups decreased significantly until the end of the experiment (P < 0.01). The 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 and DSS+ConPP groups (P < 0.005). These results indicate that intraperitoneal administration of SFRP2 functional peptides can inhibit the weight loss trend in mice with experimental colitis.

[0132] Using the normal control group as a reference (considered as 0), the disease activity index calculated for the four groups of mice was plotted as a line graph (Figure 10B, Table 12). The line graph showed that starting from day 4, 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 statistically significant difference in the DAI between the SFRP2 functional polypeptide group and the normal control group (P > 0.05).

[0133] On the seventh day after starting DSS drinking, mice were sacrificed, and their colons, mesenteric lymph nodes, serum, and feces were collected. The colons of the four groups of mice were photographed overhead (Figure 10C). As can be seen, the colons of mice with DSS-induced experimental colitis were significantly shortened. The colon length of the ConPP-treated group was close to that of the DSS+PBS group. The colon of the SFRP2 functional polypeptide-treated group was longer than that of the DSS+PBS group, but still slightly shorter than that of the normal control group. Statistical analysis of the colon lengths of the four groups of mice was performed and a bar graph was plotted (Figure 10D, Table 13). The results showed that the colon lengths of mice in the DSS+PBS and DSS+ConPP groups were significantly shorter than those in the normal control group (P<0.01, P<0.005) and the DSS+SFRP2-PP96 group (P<0.01, P<0.005). There was no statistically significant difference between the normal control group and the DSS+SFRP2-PP96 group (P>0.05).

[0134] Next, the colon tissues of the four groups of mice were subjected to pathological H&E staining (E in Figure 10). The results showed that the colon epithelial structure of the mice in the normal control group 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 mice in the DSS+ConPP group did not show obvious reduction in inflammatory infiltration. In contrast, the degree of mucosal damage in the colon tissue of the mice in the SFRP2-PP96 treatment group was reduced, the mucosa or muscular layer was basically intact, the number of goblet cell loss was reduced, and the infiltration of inflammatory cells was significantly reduced.

[0135] The H&E-stained sections were scored histologically (F in Figure 10 , Table 13). The colon histopathological 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 histopathological score of the mice in the DSS+recombinant SFRP2 protein group was alleviated (P < 0.01).

[0136] Table 11

[0137] Table 12

[0138] Table 13

[0139] Example 5 SFRP2-PP96 inhibits Th17 proliferation and promotes Treg proliferation in colon tissue

[0140] Mesenteric lymph nodes were collected from each group of mice. After treatment with cell stimulants (plus protein transport inhibitors, Invitrogen) for 5 hours, cells were stained for CD4 surface markers and IL-17A intracellularly. Alternatively, cells were directly stained for CD4 and CD25 surface markers and the nuclear transcription factor Foxp3. Flow cytometry analysis was performed, and the proportions of CD4+IL-17A+Th17 and CD4+CD25+Foxp3+Treg subsets were analyzed using Flowjo 10.4.0 software, after sequentially gating lymphocytes, removing adherent cells, and removing dead cells. The results showed that compared with the normal control group, the proportion of Th17 subsets in the mesenteric lymph nodes of mice with DSS-induced colitis was increased (P < 0.01). Treatment with the SFRP2 functional peptide SFRP2-PP96 decreased the proportion of Th17 subsets (P < 0.01), with statistically significant differences (Figure 11, AB, and Table 14). In addition, compared with the normal control group, the proportion of Treg subsets in the mesenteric lymph nodes of mice with DSS-induced colitis was reduced (P < 0.05), and the proportion of Treg subsets increased after treatment with the SFRP2 functional peptide SFRP2-PP96 (P < 0.05), with statistically significant differences (Figure 11, CD, Table 14). ConPP treatment had no significant effect on the proportions of either Th17 or Treg subsets (P > 0.05).

[0141] To further explore the effect of SFRP2 functional polypeptide on the number of Th17 cells and Treg cells in colon tissue, immunohistochemical staining for RORγt and FOXP3 was performed on paraffin sections of colon tissue from each group of mice. The results showed that the number of RORγt-positive cells in the colon tissue of the normal control group was relatively low, while 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). 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 significantly less than that of the DSS+PBS group and the DSS+ConPP group (P < 0.01, Figure 11E and F, Table 15). 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, Figure 11E and G, Table 15).

[0142] Table 14

[0143] Table 15

[0144] Example 6 SFRP2-PP96 inhibits the expression of pro-inflammatory proteins and promotes the expression of anti-inflammatory proteins in inflamed colon tissue

[0145] A section of colon tissue was collected from each group of mice for inflammation-related protein microarray analysis. A total of 18 protein expression levels were measured.

[0146] Further quantitative analysis revealed significant differences in the expression of four proteins in colonic tissue between the groups: IL-17A, IL-22, IL-10, and TGF-β1. Histograms revealed that the expression levels of the proinflammatory proteins IL-17A and IL-22 in the colonic tissue of mice with colitis were significantly elevated compared with those in the normal control group (P < 0.05, Figure 12A and B, Table 16). ConPP treatment did not significantly decrease the expression levels of IL-17A and IL-22 (P > 0.05, Figure 12A and B, Table 16). However, SFRP2-PP96 treatment significantly decreased their expression levels (P < 0.05, Figure 12A and B, Table 16). In addition, the expression levels of anti-inflammatory proteins IL-10 and TGF-β1 in the colon tissue of colitis mice were significantly lower than those in the normal control group (P < 0.05, P < 0.005, C and D shown in Figure 12, Table 16), and the control peptide ConPP treatment had no significant effect on their expression levels (P > 0.05, C and D shown in Figure 12, Table 16). However, after treatment with the SFRP2 functional peptide SFRP2-PP96, the expression levels of IL-10 and TGF-β1 were significantly increased (P < 0.05, P < 0.01, C and D shown in Figure 12, Table 16).

[0147] Table 16

[0148] Example 7 SFRP2-PP96 may promote colitis recovery by regulating the proportion of intestinal flora

[0149] Intestinal contents of each group of mice were analyzed by 16S rDNA sequencing and data integration. Fecal microbiota of each group of mice were analyzed at the phylum and genus levels. As shown in Figure 13A, the numbers of Firmicutes (orange) and Bacteroidetes (blue) decreased in the DSS+PBS and DSS+ConPP groups compared with the control group, and the Firmicutes / Bacteroidetes ratio also decreased (p < 0.05, Figure 13B, Table 17). In the DSS+SFRP2-PP96 group, the numbers of Firmicutes (orange) and Bacteroidetes (blue) decreased compared with the control group, but the Firmicutes / Bacteroidetes ratio increased to a level close to that of the control group and was significantly higher than that of the DSS+PBS and DSS+ConPP groups (p < 0.01, p < 0.05, Figure 13B, Table 17), indicating that treatment with SFRP2 functional peptide can restore the balance of some intestinal flora.

[0150] At the genus level, the predominant bacteria in the normal control group were Muribaculaceae. The relative abundance of Muribaculaceae was lower in the DSS+PBS group than in the normal control group (P < 0.005, Figure 13C and D, Table 17). ConPP treatment did not significantly alter the relative abundance of Muribaculaceae, but SFRP2-PP96 treatment significantly increased the relative abundance of Muribaculaceae (P < 0.01, Figure 13C and D, Table 17). The trend of changes in the Lactobacillus (Lachnospiraceae) genus across the four groups of mice was similar to that of the Muribaculaceae (P < 0.01, Figure 13C and E, Table 17). In the DSS+SFRP2-PP96 group, Dubosiella became the dominant bacteria, significantly higher than in the other two groups (P < 0.05, P < 0.005, Figure 13C and F, Table 17).

[0151] The above results prove that SFRP2-PP96 helps to improve intestinal flora imbalance and promote the recovery of enteritis.

[0152] Table 17

[0153] Example 8 SFRP2-PP96 treatment of experimental periodontitis in mice

[0154] Three weeks after SFRP2 functional peptide injection, mice were sacrificed and bilateral maxillae were removed. Palatal soft tissue was excised for RNA extraction. The maxillary tissue was fixed with 4% paraformaldehyde and then imaged with micro-CT. The therapeutic effect of the treatment on experimental periodontitis in mice was evaluated by imaging.

[0155] The 3D imaging results are shown in Figure 14. The height from the cementoenamel junction to the alveolar crest, i.e., clinical attachment loss (AL, CEJ-AB), was measured. The results, as shown in Table 18, showed that compared with the normal control group, the CEJ-AB of mice in the NaCl control and ConPP treatment groups was significantly increased (P < 0.01), while the CEJ-AB in the SFRP2-PP96 treatment group was restored (P < 0.05).

[0156] Table 18

[0157] Example 9 SFRP2-PP96 promotes the odontogenic differentiation of DPSCs in vitro and partially rescues the odontogenic differentiation of SFRP2sh DPSCs

[0158] 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.

[0159] 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 cultured at 1×10 5 The cells were seeded 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.

[0160] On day 7 of odontogenesis induction, alkaline phosphatase activity in DPSCs was measured using an ALP activity kit (Sigma-Aldrich, USA). Optical density (OD) was measured using a microplate reader (absorbance at 405 nm), and ALP activity was calculated using a standard ALP activity curve. The results showed that ConPP had no significant effect on ALP activity in DPSCs (P>0.05, Figure 17A), while SFRP2-96 peptide significantly enhanced ALP activity in DPSCs (P<0.01, Figure 15A).

[0161] After two weeks of odontogenesis induction, cells were fixed with tissue fixative (Biyuntian) for 1 hour and stained with 2% Alizarin Red (Sigma-Aldrich, USA). Calcium ion concentration was also measured: Alizarin Red dye was decolorized with 10% cetylpyridinium chloride (dissolved in 10 mmol / L sodium phosphate) at 20°C ± 5°C for 30 minutes. OD values ​​were measured using a microplate reader (absorbance at 562 nm), and calcium ion concentrations in the samples were calculated using a standard calcium ion concentration curve. The results showed that ConPP had no significant effect on the Alizarin Red staining intensity and calcium ion concentration of DPSCs compared with the control group (P>0.05, Figures 15B-15C), while SFRP2-96 peptide significantly enhanced Alizarin Red staining intensity and calcium ion concentration in DPSCs (P<0.01, Figures 15B-15C).

[0162] On day 7 of odontogenesis induction, intracellular proteins were extracted and Western blot analysis was performed to determine the levels of intracellular DSPP and DMP1 proteins. The results showed that ConPP had no significant effect on the levels of intracellular DSPP and DMP1 proteins in DPSCs (Figure 15D), whereas SFRP2-96 peptide significantly increased their levels (Figure 15D).

[0163] 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.

[0164] Short hairpin RNAs (shRNAs) complementary to the target gene SFRP2 were subcloned into the pLKO.1 lentiviral vector (Addgene, USA). Scrambled shRNAs (Scramsh) served as a knockdown control virus. The target sequence of the SFRP2 shRNAs was as follows: 5'-ttgatgtaggttatctccttc-3'. DPSCs were transfected with scrambled and SFRP2 shRNAs, and the efficiency of SFRP2 knockdown was assessed by real-time RT-PCR. Results showed that SFRP2 RNA expression was significantly reduced in the SFRP2sh group (P < 0.01, Figure 16A). Stably transfected DPSCs were then induced into odontogenesis, and 10 μg / mL of SFRP2-PP96 was added to the SFRP2sh group. On day 5 of odontogenesis induction, ALP activity was measured, revealing a significant decrease in ALP activity in the SFRP2sh group compared with the Scramsh group (P < 0.01, Figure 16B), but this activity was partially rescued by SFRP2-PP96 (P < 0.05, Figure 16B). Similarly, at week 2 of odontogenesis induction, Alizarin Red staining results showed a significant decrease in the depth of Alizarin Red staining in the SFRP2sh group compared with the Scramsh group (Figure 16C), but this was partially rescued by SFRP2-PP96 (Figure 16C). Furthermore, protein analysis at day 7 of odontogenesis induction revealed a decrease in the expression of intracellular DSPP and DMP1 proteins after SFRP2 knockout (Figure 16D), but this was partially rescued by SFRP2-PP96 (Figure 16D).

[0165] Example 10 SFRP2-PP96 promotes DPSCs-mediated tooth regeneration in the jaw

[0166] To investigate the effect of SFRP2-PP96 on DPSCs-mediated tooth regeneration in the mandible, the right maxillary and mandibular incisors of rabbits were extracted, and a mixture of 10 μg / ml of ConPP or SFRP2-PP96 cells and hydrogel scaffolds was reimplanted and sutured to establish a rabbit mandibular tooth regeneration model ( FIG17 ).

[0167] Three months later, the animals were sacrificed, and microCT scans were taken to measure the volume of regenerated high-density calcifications within the jaw. The results showed that ConPP had no significant effect on the volume of regenerated high-density calcifications within the jaw compared with the control group (P>0.05, Figures 18A and 18B), while the volume of regenerated high-density calcifications within the jaw in the SFRP2-PP96-treated group was significantly increased (P<0.05, Figures 18A and 18B). Gross observation of jaw cross-sections revealed no significant differences between the ConPP and control groups, while the SFRP2-96 peptide-treated group formed significantly more high-density calcifications than the control group (Figure 18C).

[0168] Further histological staining of rabbit jaw sections revealed that there was no significant difference between the ConPP group and the control group, while the SFRP2-96 polypeptide-treated group formed a large number of dentin-like regenerations surrounded by a large number of odontoblast-like cells (Figure 18D). Masson staining also showed that there was no significant difference between the ConPP group and the control group, but the SFRP2-96 polypeptide-treated group formed more collagen and osteoid (Figure 18E). In addition, immunohistochemistry and immunofluorescence staining showed that the expression of DSPP and DMP1 in the jaws of the SFRP2-96 polypeptide-treated group was stronger (Figure 18F, Figure 18G), while there was no significant difference between the ConPP group and the control group (Figure 18F, Figure 18G).

[0169] Example 11 SFRP2-PP96 inhibits the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a

[0170] To investigate the binding ability of SFRP2-PP96 to Wnt3a, a microscale thermophoresis (MST) assay was performed. Wnt3a recombinant protein and SFRP2-PP96 were placed in a capillary tube at a concentration gradient. Infrared laser heating generated a microscopic temperature gradient, causing thermophoresis and shifts in the fluorescence distribution within the reaction system. The results showed a Kd value of 8.723E-6, indicating that SFRP2-PP96 binds to Wnt3a in vitro (Figure 19A).

[0171] 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 verified by Western blot experiments. The results showed that the total intracellular β-catenin was different among the three groups, but SFRP2-PP96 enhanced the intracellular p-β-catenin level and inhibited the Wnt / β-catenin signaling pathway (Figure 19B). ConPP had no significant effect on the intracellular p-β-catenin level (Figure 19B). In addition, the intracellular p-β-catenin level decreased after SFRP2 knockout, but the intracellular p-β-catenin level increased after the addition of SFRP2-PP96 (Figure 19C).

[0172] Therefore, it was concluded that SFRP2-PP96 inhibited the intracellular Wnt / β-catenin signaling pathway by binding to Wnt3a.

[0173] The above describes in detail the SFRP2 functional polypeptides and their applications provided by the present invention. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above examples is only intended to help understand the methods and core concepts of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0174] Sequence Listing

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.

11. A method for preventing and / or treating periodontitis, characterized in that: Administer any of the following: (I), the SFRP2 polypeptide according to claim 1; (II), the medicament according to claim 9; or (III) The pharmaceutical combination according to claim 10.

12. A method for preventing and / or treating colitis, characterized in that: Administer any of the following: (I), the SFRP2 polypeptide according to claim 1; (II), the medicament according to claim 9; or (III) The pharmaceutical combination according to claim 10.

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