Composition containing pi15 protein and its application in differentiation regulation of mesenchymal cells related to tooth development

Abstract

The application provides a composition containing a PI15 protein and its application in differentiation regulation of mesenchymal cells related to tooth development. Compared with the prior art, the application has the following advantages: high specificity and low off-target effect in the scenario of tooth development are realized; the target mechanism is innovative, and the regulation efficiency is significantly improved; a physiological simulation regulation system of 'epithelial-mesenchymal interaction' is first constructed; a new path for tooth tissue regeneration and clinical transformation is opened; the technology breaks through the limitations of traditional general inhibitors in clinical application, and lays a solid technical foundation for the development of a new generation of precise regeneration therapy.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to compositions containing PI15 protein and their application in the regulation of tooth development-related mesenchymal cell differentiation. Background Technology

[0002] The WNT signaling pathway is a core signaling pathway regulating mesenchymal cell morphogenesis and directed differentiation. Its activity balance directly determines the differentiation fate of mesenchymal cells into functional cells such as odontoblasts and osteoblasts, thus affecting physiological processes such as tooth germ development and tooth tissue repair and regeneration. Currently, known WNT pathway inhibitors can participate in regulating mesenchymal cell morphogenesis and differentiation; however, most existing WNT pathway inhibitors are non-specific, lacking tissue or cell specificity. When regulating mesenchymal cell function, these inhibitors easily interfere with the WNT pathway activity of other tissues and organs, leading to off-target effects and failing to accurately meet the regulatory needs of mesenchymal cells in specific scenarios such as tooth development. Furthermore, existing WNT pathway inhibitors mostly target downstream molecules in the pathway, resulting in limited regulatory efficiency and difficulty in combining with epithelial-mesenchymal interactions to function, failing to meet the precise regulatory needs of "epithelial-derived signaling molecules regulating mesenchymal cell function" in physiological processes such as tooth development. Therefore, there is an urgent need for a tissue-specific molecular target and application strategy capable of precisely regulating mesenchymal cell function. Summary of the Invention

[0003] To address the aforementioned technical limitations, this application proposes a composition containing PI15 protein and its application in the regulation of tooth development-related mesenchymal cell differentiation; it overcomes the deficiencies and defects mentioned in the background art.

[0004] To achieve the above objectives, this application adopts the following technical solution: The inventive point of this application is to provide an inhibitor of the WNT signaling pathway, wherein the inhibitor includes the PI15 protein or its encoding gene.

[0005] Optionally, in the above-mentioned inhibitor, the PI15 protein is a secretory protein derived from dental epithelium, and its amino acid sequence is shown in SEQ ID No. 1.

[0006] SEQ ID No. 1: MIMNSAVSLVILLSLLCEAHTVVLLNPTDSSLPANNFTDTEAALSTPLESADIPKARRKRYISQNDMIAILDYHNQVRGKVFPPAANMEYMVWDENLAKSAEAWAATCIWDHGPSYLLRFLGQNLSVRT GRYRSILQLVKPWYDEVKDYAFPYPQDCNPRCPMRCFGPMCTHYTQMVWATSNRIGCAIHTCQNMNVWGSVWRRAVYLVCNYAPKGNWIGEAPYKVGVPCSSCPPSYGGACTDNLCFPGVTTNYLYWFK.

[0007] Optionally, the above-mentioned inhibitor further includes SFRP1 protein or its encoding gene; the SFRP1 protein is a secretory protein derived from dental papilla mesenchyme, and its amino acid sequence is shown in SEQ ID No. 2.

[0008] SEQ ID No. 2: MGVGRSARGRGGAASGVLLALAAALLAAGSASEYDYVSFQSDIGSYQSGRFYTKPPQCVDIPVDLRLCHNVGYKKMVLPNLLEHETMAEVKQQASSWVPLLNKNCHMGTQVFLCSLFAPVCLDRPIYPCRWLCEAVRDSCEPVMQFFGFYWPEMLKC DKFPEGDVCIAMTPPNTTEASKPQGTTVCPPCDNELKSEAIIEHLCASEFALRMKIKEVKKENGDKKIVPKKKKPLKLGPIKKKELKRLVLFLKNGADCPCHQLDNLSHNFLIMGRKVKSQYLLTAIHKWDKKNKEFKNFMKRMKNHECPTFQSVFK.

[0009] The second inventive point of this application is to provide the application of the above-mentioned WNT signaling pathway inhibitor in the preparation of any of the following products: 1) Products used to regulate mesenchymal cell morphogenesis and differentiation; 2) Products used to promote dentin formation or tooth tissue regeneration.

[0010] Optionally, in the above application, the mesenchymal cells are dental papilla mesenchymal cells.

[0011] Optionally, in the above-described application, the product for regulating mesenchymal cell morphogenesis and differentiation achieves its regulatory effect by applying an effective dose of WNT signaling pathway inhibitor to mesenchymal cells, negatively regulating the expression of WNT pathway activation-related molecules Axin2, Ccnd1, and / or β-catenin in the cells, thereby inhibiting the differentiation of mesenchymal cells into odontoblasts or osteoblasts.

[0012] Optionally, in the above-described application, the product for regulating mesenchymal cell morphogenesis and differentiation works by knocking down or inhibiting the expression of PI15 protein or its encoding gene and SFRP1 protein or its encoding gene in the microenvironment of mesenchymal cells, thereby relieving the inhibition of the WNT signaling pathway in mesenchymal cells, promoting the expression of odontoblast differentiation markers Dlx5, Runx2 and / or MMP7 in mesenchymal cells, and thus promoting the differentiation of mesenchymal cells into odontoblasts to achieve the regulatory effect.

[0013] Optionally, in the above-described application, the product for promoting dentin formation or tooth tissue regeneration works by reducing the concentration or activity of PI15 / SFRP1 protein at the target site, thereby relieving the negative inhibition of the WNT pathway of dental papilla mesenchymal cells by PI15 / SFRP1, thus promoting odontoblastic differentiation of dental papilla mesenchymal cells and achieving the function of promoting dentin formation or tooth tissue regeneration.

[0014] The third inventive point of this application is to provide a pharmaceutical composition for promoting dentin formation or tooth tissue regeneration, the pharmaceutical composition comprising PI15 protein or recombinant PI15 protein, and / or SFRP1 protein or recombinant SFRP1 protein; the concentration ratio of PI15 protein to SFRP1 protein is selected as 1:1, each 50 ng / mL.

[0015] Optionally, the above-described pharmaceutical composition is used to treat dentin defects, pulp injury, or tooth developmental abnormalities.

[0016] Compared with the prior art, this application has the following advantages: 1. Achieved high specificity and low off-target effects in the context of tooth development: The WNT signaling pathway inhibitor (PI15 protein) provided in this application is specifically and highly expressed in dental epithelium, enabling precise targeting of the tooth development microenvironment. This tissue specificity not only ensures precise regulation of target mesenchymal cells but also minimizes interference with non-target tissues, solving the key challenge of existing technologies being unable to adapt to specific physiological scenarios such as tooth development, and significantly improving the safety and precision of regulation.

[0017] 2. Innovative target mechanism significantly improves regulatory efficiency: PI15 acts on key regulatory nodes of the WNT pathway. By directly intervening in the core activity of the pathway, PI15 achieves more efficient and thorough inhibition of the signaling pathway. This upstream or key node intervention mechanism makes the regulation of mesenchymal cell morphogenesis and differentiation more rapid and stable, overcoming the insufficient efficacy of traditional inhibitors due to their limited site of action.

[0018] 3. For the first time, a physiological simulation and regulatory system for "epithelial-mesenchymal interaction" was constructed: This application is the first to disclose and utilize a paracrine mechanism mediated by an epithelial-derived secretory protein (PI15). During tooth development, the interaction between the epithelium and the mesenchyme is central to determining organ morphogenesis. This patented technology simulates the natural biological process of "epithelial regulation of the mesenchyme" through PI15, perfectly matching the intrinsic logic of tooth development. This not only fills the gap in the field of epithelial-specific WNT inhibitors but also provides a novel molecular tool and theoretical basis for studying epithelial-mesenchymal interactions.

[0019] 4. It has opened up a new pathway for tooth tissue regeneration and clinical translation: By overcoming the two major bottlenecks of specificity and physiological adaptability, this patented technology offers a highly promising application solution for dental tissue engineering and regenerative medicine. Utilizing PI15 for "precise regulation mediated by epithelial-derived signaling molecules" can effectively guide the directed differentiation and tissue construction of mesenchymal cells, which has significant translational value for clinical treatments such as in-situ tooth regeneration and periodontal tissue repair. This technology overcomes the limitations of traditional universal inhibitors in clinical applications, laying a solid technical foundation for the development of next-generation precision regenerative therapies. Attached Figure Description

[0020] Figures 1-4 The transcriptome is shown as a transcriptomic profile analyzed by single-cell transcriptomics sequencing from the budding to bell stages of the tooth germ; among which, Figure 1 The results are shown as single-cell transcriptomic sequencing of mouse E13.5 budding stage, E15.5 capping stage and E17.5 bell-shaped late tooth germ. Figure 2 The results are shown in the figure, which represents the identification based on cell population characteristics. Figure 3 The results are shown as a graph illustrating the cell proportion analysis for each population. Figure 4 The graph shows the analysis results of the top three specifically highly expressed molecules in each cell population; Figures 5-10 The figure shows the results of molecular expression characterization analysis of tooth germ epithelium and mesenchyme; Figures 5-6 The analysis results show the lineage differentiation characteristics of tooth germ epithelium; Figure 5 The tooth germ epithelium was regrouped, and ameloblasts, inner enamel epithelium, outer enamel epithelium, stellate interlaminar layer, and lamina neck rings were identified. A pseudo-time series analysis was then performed.

[0021] Figure 6 The display shows specific genes for the regrouping of embryonic epithelium. Genes specifically expressed in the outer enamel epithelium are GNRH1 and SOSTDC1, while those in the inner enamel epithelium highly express SHH and CYP26A1. The stellate reticulate intermediate layer highly expresses S100A6 and RHOV.

[0022] Figures 7-9 The analysis results show the lineage differentiation characteristics of dental papilla mesenchyme; Figure 7 The data shows that the mesenchyme of the dental papilla was regrouped, and the coronal and radicular dental papilla cell populations were identified and subjected to pseudo-time series analysis.

[0023] Figure 8 The genes highly expressed in the coronal papilla are OMD, DKK1, and HAPLN1, while those highly expressed in the radicular papilla are VIM, CRABP1, and SFRP1.

[0024] Figure 9 The analysis shows the high expression of secretory proteins in the coronal mesenchyme of the dental papilla and the high expression of secretory proteins in the inner enamel epithelium. A total of 515 secretory proteins were expressed by both the inner enamel epithelium and the dental papilla. Among them, the inner enamel epithelium expressed 1014 proteins in a higher manner than the dental papilla, and the coronal dental papilla expressed 156 proteins in a higher manner than the inner enamel epithelium.

[0025] Figure 10 The results show the expression profiles of secretory proteins in the mesenchyme of the inner enamel epithelium and the coronal papillae. Figures 11-14 The results are shown as PI15 staining validation and preliminary functional analysis. Figure 11 The results of PI15 immunofluorescence staining are shown for mouse tooth germs and miniature pig tooth germs. Figure 11 A shows the results of PI15 immunofluorescence staining of mouse tooth germs E13.5 and E17.5; Figure 11 B represents the results of PI15 immunofluorescence staining of tooth germs E40 and E50 from miniature pigs. Figure 12 The results are shown as molecular spectroscopic analysis of the interaction with PI15; Figure 13 The image shows the results of differentiation and WNT pathway activation after separating mouse tooth germ epithelium and mesenchyme, knocking down and overexpressing PI15 in epithelial cells and co-culturing them with mesenchymal cells. Figure 14 Displayed as Figure 13 Statistical results of expression levels of differentiation markers and WNT pathway activating molecules in the PI15 knockdown group and the IWR-1 group, a classic inhibitor of the WNT pathway; Figure 15 The results show the in vivo and in vitro expression patterns and functional validation of PI15 in tooth germ development. Figure 15 Figure A shows the statistical results of mRNA expression levels obtained after isolating the tooth germ epithelium and mesenchyme of mice E13.5, E15.5, E17.5 and PN0, detecting the expression pattern of PI15 in the tooth epithelium and the activation status of the WNT pathway in the tooth papilla mesenchyme. Figure 15 B represents the mRNA expression level statistics obtained by adding different concentrations of PI15 recombinant protein to the primary cultured dental papilla mesenchyme and detecting the activation and differentiation status of the WNT pathway.

[0026] Figures 16-17 The graph shows the results of PI15 functional verification using a mouse in vivo open-medullary model. Figure 16 The illustration shows the ability of dental papilla mesenchyme co-cultured under different conditions to differentiate into dentin by introducing it into an open pulp chamber model (including PI15 overexpression group, SFRP1 addition group, PI15 knockdown group, and PI15 and SFRP1 combined addition group). Figure 16 A is the experimental flowchart for importing the medullary canal opening model. Figure 16 B shows the results of the differentiation into dentin capacity verified by the open pulp chamber model; Figure 17 The figure shown is a result of the mechanism verification of PI15 using an in vivo medullary canal model. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0029] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0030] Example 1 I. Single-cell transcriptomics sequencing analysis of the transcriptomic profile from the budding to bell stages of the tooth germ: This experiment selected mouse tooth germs at different developmental stages for single-cell transcriptome sequencing and related analysis. The specific procedure is as follows: (A) Sample Selection and Sequencing: Mouse tooth germ tissues from the E13.5 budding stage, E15.5 capping stage, and E17.5 bell-shaped stage were selected, with three biological replicates for each group. Tooth germs were isolated under aseptic conditions, and surrounding connective tissue was removed. Single-cell suspensions were obtained by digestion with collagenase and neutral protease. Cell clumps were removed by filtration through a 70μm filter. After treatment with erythrocyte lysis buffer, cell viability was verified to be ≥90% by trypan blue staining. Single-cell sequencing libraries were constructed using the 10× Genomics Chromium platform. After quality control, high-throughput sequencing was performed using the Illumina sequencing platform to obtain raw single-cell transcriptome data for tooth germs at the three developmental stages.

[0031] (B) Cell Cluster Identification: The raw sequencing data was filtered, standardized, and batch-corrected to remove low-quality cells and impurities. PCA dimensionality reduction and t-SNE clustering analysis were performed using Seurat software to obtain different cell clusters. Based on known cell markers related to tooth germ development (such as CK14 for dental epithelial cells and VIM for dental mesenchymal cells) and the gene expression characteristics of each cell cluster, each cell cluster was precisely identified to determine its corresponding cell type (e.g., dental epithelial cells, ameloblastic precursor cells, dental mesenchymal cells, etc.).

[0032] (C) Cell population ratio analysis: Based on the identified cell clusters, the proportion of each cell population in the three developmental stages (E13.5, E15.5, E17.5) was statistically analyzed, and a cell ratio statistical chart was drawn to analyze the trend of the proportion of each cell population in different developmental stages and clarify the dynamic evolution characteristics of cell subpopulations during tooth germ development.

[0033] (D) Analysis of Specific Highly Expressed Molecules: Differential gene expression analysis was performed on each identified cell population to screen out molecules that are specifically highly expressed in each cell population. The top three specifically highly expressed molecules in each cell population were selected for summary analysis to clarify the molecular marker characteristics of each cell population.

[0034] II. Analysis of the molecular expression characteristics of tooth germ epithelium and mesenchyme: Based on single-cell transcriptome sequencing data of mouse tooth germs at E13.5 budding stage, E15.5 capping stage, and E17.5 bell-shaped late stage, this study focuses on analyzing the differentiation characteristics of the tooth germ epithelium and dental papilla mesenchymal lineage and the expression of key secreted proteins. The specific workflow is as follows: (A-B') Analysis of lineage differentiation characteristics of tooth germ epithelium: Tooth germ epithelium-related cell populations were screened from the identified cell clusters, and pseudo-time series analysis trajectories were constructed based on single-cell transcriptome data to clarify the lineage differentiation pathway of tooth germ epithelial cells from primitive epithelial cells to ameloblastic precursor cells and inner enamel epithelial cells; (CE) Analysis of the lineage differentiation characteristics of dental papilla mesenchymal cells: related cell clusters of dental papilla mesenchymal cells were screened out, and pseudo-temporal analysis and cell trajectory clustering methods were used to analyze the lineage differentiation process of dental papilla mesenchymal cells into odontoblast precursor cells and dental follicle cells. (F) Expression profile analysis of secretory proteins in the inner enamel epithelium and coronal papilla mesenchymal cell clusters: Gene expression matrices were extracted from the inner enamel epithelial cell clusters and the coronal papilla mesenchymal cell clusters, respectively. Genes encoding secretory proteins were screened in the two cell types to construct secretory protein expression profiles. Differential analysis and enrichment analysis were performed on the expression profiles to screen secretory proteins that were specifically highly expressed in the two cell types. After analysis, it was found that PI15 secretory protein was expressed in the tooth germ epithelium at different time points, and was particularly highly expressed in the early developmental stage.

[0035] III. PI15 staining verification and preliminary functional analysis: This experiment investigated the spatiotemporal expression characteristics of PI15 and its regulatory role in tooth germ epithelial-mesenchymal interactions and the WNT pathway through multi-species immunofluorescence staining, molecular interaction analysis, and cell co-culture experiments. The specific procedure is as follows: (A) Immunofluorescence staining of PI15 in mouse tooth germs: Mouse E13.5 and E17.5 tooth germ tissues were selected, routinely decalcified, embedded, and sectioned. After antigen retrieval and blocking, PI15-specific primary antibody was added and incubated overnight. After incubation with fluorescent secondary antibody, DAPI staining was used to label cell nuclei. The images were observed and acquired under a fluorescence microscope to analyze the expression location and intensity differences of PI15 in mouse tooth germs at different developmental stages. (B) Immunofluorescence staining of PI15 in miniature pig tooth germs: Miniature pig E40 and E50 tooth germ tissues were selected, and the above immunofluorescence staining procedure was repeated. A negative control (without primary antibody) was set up simultaneously to exclude non-specific binding. The conserved expression characteristics of PI15 in mouse and miniature pig tooth germs were compared and analyzed to provide a basis for clinical translation. (C) PI15 interaction molecular profile analysis: Based on mouse tooth germ single-cell transcriptome sequencing data and combined with PI15 expression characteristics, genes co-expressed with PI15 were screened; the STRING database and Co-IP experiments were used to verify the interacting molecules of PI15, construct the PI15 interaction molecular profile, focus on analyzing the interacting molecules related to the WNT pathway, and explore the molecular basis of PI15 regulatory pathway. (D) Validation of PI15 regulation of epithelial-mesenchymal co-culture: Mouse tooth germ epithelial and mesenchymal cells were aseptically isolated, and PI15 was knocked down and overexpressed in the epithelial cells, respectively. Four co-culture systems were constructed (blank control group, PI15 knockdown group, PI15 overexpression group and WNT3A ligand group). After culture, the expression of dental papilla mesenchymal cell differentiation markers Dlx5 and Runx2 and WNT pathway activation-related molecules Axin2, Ccnd1 and β-catenin were detected to verify the regulatory effect of PI15 on mesenchymal cell differentiation and WNT pathway activation.

[0036] IV. Verification of the in vivo and in vitro expression patterns and functions of PI15 in tooth germ development: This experiment investigated the regulatory role of PI15 on the WNT pathway and differentiation of dental papilla mesenchymal tissue through the separation and detection of tooth germ epithelium and mesenchymal tissue and the intervention experiment of primary mesenchymal cells. The materials and methods are as follows: tooth germs of C57BL / 6 mice at the E13.5, E15.5, E17.5 and PNO stages were selected. After aseptic separation, they were treated with trypsin-EDTA digestion solution. The tooth germ epithelium and dental papilla mesenchymal tissue were separated under micromanipulation. The cells were washed three times with PBS and collected by centrifugation.

[0037] (A) Detection of PI15 expression and WNT pathway activation status: Total RNA and protein were extracted from tooth germ epithelial cells and dental papilla mesenchymal cells at different developmental stages. The mRNA expression level of PI15 in dental epithelial cells was detected by qPCR to detect the PI15 protein expression pattern. At the same time, the expression of WNT pathway activation-related molecules Axin2 and β-catenin in dental papilla mesenchymal cells was detected by qPCR, as well as the mRNA levels of dental papilla mesenchymal differentiation-related molecules Runx2 and MMP7, to clarify the association between PI15 expression and WNT pathway activation status at different developmental stages. (B) Experiment on primary mesenchymal cell intervention with recombinant PI15 protein: Isolated dental papilla mesenchymal cells were seeded into culture dishes, and DMEM medium containing serum was added. The cells were then cultured in a 37℃, 5% CO2 incubator until approximately 70% confluence. The cells were then divided into a blank control group and treatment groups with different concentrations of recombinant PI15 protein (gradient concentrations of 0, 10, 50, 100, and 200 ng / mL), with three replicates per group. After 48 h of intervention culture, cells were collected, and the expression of WNT pathway molecules Axin2 and β-catenin, as well as mesenchymal differentiation markers MMP7 and Runx2, was detected using qPCR. The effects of different concentrations of PI15 on the WNT pathway activation and differentiation status of mesenchymal cells were assessed. The experiment was repeated three times to ensure the reliability of the results. V. Verification of PI15 function using a mouse in vivo myelotomy model: This experiment used an animal pulpotomy model to verify the odontoblastic differentiation capacity of the mesenchyme in the co-cultured dental papilla and to draw a diagram of the PI15 regulatory mechanism. The materials and methods are as follows: Healthy C57BL / 6 mice (8 weeks old, half male and half female) were selected and acclimatized for 1 week to establish a mouse molar pulpotomy model. The pulp cavity was exposed under aseptic conditions, the pulp tissue was removed, and the pulp was rinsed with physiological saline before use.

[0038] (AB) Implementation and Differentiation Capacity Verification of Co-cultured Dental Papillary Mesenchymal Cell Pulp Opening Model: Dental papillary mesenchymal cells were collected from co-culture systems under different conditions (blank control group, PI15 knockdown group, PI15 overexpression group, and WNT3A ligand group). After trypsin digestion and centrifugation, the cells were collected and resuspended in PBS to adjust the cell concentration to an appropriate level. Mesenchymal cells from different groups were introduced into the pulp chambers of corresponding mouse pulp opening models. The wounds were sealed with glass ionomer cement. Mice were routinely fed post-surgery. Six mice were used in each group, with three biological replicates. Mice were sacrificed at specific time points post-surgery. Molar tissue was isolated, decalcified, embedded, and sectioned. Masson staining was used to observe pulp chamber repair and verify the differences in the ability of mesenchymal cells to differentiate into dentin under different conditions. (C) Mechanism diagram: Based on all experimental results of this study, the spatiotemporal expression characteristics and interaction molecular profile of PI15 in tooth germ epithelium, as well as the regulatory role of PI15 on the activation of the WNT pathway (Axin2, Ccnd1, β-catenin) in dental papilla mesenchymal and odontoblastic differentiation, are integrated to clarify the core regulatory axis of "PI15-interacting molecules-WNT pathway-mesenchymal odontoblastic differentiation" and to intuitively present the molecular mechanism by which PI15 regulates the interaction between tooth germ epithelium and mesenchymal and the formation of dentin.

[0039] Example 3 This application focuses on a series of experiments investigating the expression characteristics, regulatory mechanisms, and functions of PI15 during tooth germ development. Through single-cell transcriptome sequencing, immunofluorescence staining, cell function verification, and animal model experiments, it was clarified that PI15, as a tooth epithelium-specific secreted protein, plays a core role in influencing the differentiation of tooth papilla mesenchyme into dentin by regulating the WNT pathway and mediating tooth germ epithelium-mesenchyma interactions. The following is a summary of the experimental results: I. Single-cell transcriptomics sequencing analysis of the transcriptomic profile from the budding to bell stages of the tooth germ: 1. Single-cell transcriptome sequencing data passed quality control: Single-cell transcriptome data were successfully obtained from mouse late tooth germs at E13.5 budding stage, E15.5 capping stage, and E17.5 bell-shaped stage. After filtering and quality control, a total of 42,531 single cells were obtained. No significant batch effect was observed at the three time points, and the data can be used for subsequent cell population and gene expression analysis. Figure 1 ).

[0040] 2. Precise identification of tooth germ cell populations: Analysis using Seurat software identified multiple cell subpopulations, including tooth germ epithelial cells, ameloblastic precursor cells, dental papilla mesenchymal cells, odontoblastic precursor cells, and dental follicle cells. Figure 2 Each cell population can be accurately distinguished by specific markers (such as epithelial CK14 and mesenchymal VIM), and the cell grouping results are consistent with the cellular composition characteristics of tooth germ development.

[0041] 3. The proportion of cell populations changes dynamically during tooth germ development: E13.5 In the budding stage, primitive dental epithelial cells and undifferentiated mesenchymal cells predominate; in the cap stage, the proportion of ameloblastic and odontoblastic precursor cells increases significantly; in the bell stage, functional precursor cells further differentiate, and the proportion of ameloblastic and odontoblastic precursor cells reaches its peak, while the proportion of dental follicle cells increases synchronously, showing the cell lineage differentiation pattern of tooth germ development. Figure 3 ).

[0042] 4. Each cell population exhibits highly expressed molecular markers specific to these markers: For each identified cell subpopulation, the top three specifically highly expressed molecules were screened out. Figure 4 Dental epithelial cells, amelioblastic precursor cells, dental papilla mesenchymal cells, and odontoblastic precursor cells all have their own specific molecular markers, providing a molecular basis for the precise sorting and functional study of subsequent tooth germ epithelial-mesenchymal cells.

[0043] II. Molecular Expression Characteristics Analysis of Tooth germ Epithelium and Mesenchyme 1. The tooth germ epithelium exhibits a clear lineage differentiation trajectory: Pseudo-temporal analysis showed that tooth germ epithelial cells from E13.5 to E17.5 could differentiate in a directed manner along the pathway of "primitive epithelial cells → inner enamel epithelial cells → ameloblastic precursor cells," with a gradual upregulation of ameloblastic-related genes during the differentiation process, exhibiting continuous lineage differentiation characteristics. Figure 5 , Figure 6 ).

[0044] 2. Dental papilla mesenchyme exhibits lineage characteristics of directed differentiation: Pseudo-chronological analysis of dental papilla mesenchymal cells clarified their differentiation trajectory as "undifferentiated mesenchymal cells → coronal odontoblast precursor cells → radicular odontoblast precursor cells," with odontoblast-related gene expression gradually increasing during differentiation, matching the morphological changes in tooth germ development. Figures 7-9 ).

[0045] 3. Significant differences were found in the expression profiles of secretory proteins in the mesenchymal tissue of the inner enamel epithelium and the coronal papilla: The expression profiles of secretory proteins in two cell types were successfully constructed. Differential analysis showed that several secretory proteins were specifically overexpressed in inner enamel epithelial cells. Among them, PI15 was expressed in tooth germ epithelium at different developmental stages, and its expression level was significantly increased in early developmental stages such as E13.5 and E15.5, making it a secretory protein specifically overexpressed in tooth epithelium. Figure 10 This became the core target for subsequent research.

[0046] III. PI15 staining verification and preliminary functional analysis: 1. PI15 is expressed in mouse tooth germs with spatiotemporal specificity: Immunofluorescence staining results showed that PI15 was mainly located in the inner enamel epithelial cells of mouse E13.5 and E17.5 tooth germs. The fluorescence signal of PI15 was stronger in the epithelium of the E13.5 budding stage tooth germ, while in the E17.5 bell-shaped late stage, it was mainly concentrated at the interface between the inner enamel epithelium and the dental papilla mesenchyme, suggesting that PI15 may be involved in epithelial-mesenchymal signaling. Figure 11 A).

[0047] 2. PI15 is conserved in mouse and miniature pig tooth germs: Immunofluorescence staining results of E40 and E50 tooth germs from miniature pigs showed that PI15 was also localized in tooth germ epithelial cells and significantly expressed at the epithelial-mesenchymal interface, highly consistent with the expression characteristics of mouse tooth germs. This confirms that the expression of PI15 in tooth germ development is species-conserved, providing a basis for its clinical translational research. Figure 11 B).

[0048] 3. Successfully constructed the PI15 interaction molecular profile and screened out WNT pathway-related interacting molecules: Through co-expression analysis and validation using the STRING database and Co-IP experiments, several molecules interacting with PI15 were screened. Figure 12 This study included several key regulatory molecules of the WNT pathway, confirming that PI15 can form an interaction network by binding with WNT pathway molecules, providing a molecular basis for PI15 to regulate the WNT pathway.

[0049] 4. PI15 regulates the differentiation of dental papilla mesenchymal cells and the activation status of the WNT pathway: Epithelial-mesenchymal co-culture experiments showed that, compared with the PI15 knockdown group, the expression of differentiation markers Dlx5 and Runx2 in dental papilla mesenchymal cells was significantly downregulated in the PI15 overexpression group, and the expression of WNT pathway activating molecules Axin2, Ccnd1, and β-catenin was also significantly reduced; the WNT3A ligand group could reverse the inhibitory effect of PI15 overexpression, confirming that PI15 can negatively regulate the overactivation of the WNT pathway in dental papilla mesenchymal cells. Figure 13 , Figure 14 ).

[0050] IV. In vivo and in vitro expression patterns and functional verification of PI15 in tooth germ development: 1. PI15 expression is negatively correlated with activation of the WNT pathway in dental papilla mesenchyme: The results of tooth germ examination at mouse stages E13.5, E15.5, E17.5 and PN0 showed that ( Figure 14 The expression levels of PI15 mRNA and protein in the dental epithelium showed a gradual decreasing trend from E13.5 to E17.5, while the expression levels of WNT pathway molecules Axin2 and β-catenin, as well as differentiation markers Runx2 and MMP7 in the dental papilla mesenchyme showed a gradual increasing trend. The expression trends of the two showed a significant negative correlation, further confirming the regulatory relationship between PI15 and WNT pathway activation and mesenchymal differentiation.

[0051] 2. Recombinant PI15 protein inhibits the activation and differentiation of the WNT pathway in dental papilla mesenchyme in a concentration-dependent manner: Primary dental papilla mesenchymal cell intervention experiments showed that ( Figure 15 As the concentration of recombinant PI15 protein increased (0, 10, 50, 100, 200 ng / mL), the mRNA expression levels of WNT pathway molecules Axin2 and β-catenin, as well as differentiation markers MMP7 and Runx2, in cells gradually decreased. The inhibitory effects were significant in the 100 and 200 ng / mL concentration groups, confirming that the regulation of mesenchymal cells by PI15 is concentration-dependent.

[0052] V. Verification of PI15 function using a mouse in vivo myelotomy model: 1. PI15 negatively regulates the odontoblast differentiation capacity of dental papilla mesenchymal cells in vivo: The results of the mouse molar pulpotomy model experiment showed that ( Figure 16Masson staining results showed that, compared with the blank control group, the PI15 overexpression group and the SFRP1 addition group had increased dentin-like tissue formation in the pulp cavity repair area, indicating better repair effects; the PI15 knockdown group had decreased dentin-like tissue formation in the pulp cavity, indicating poorer repair effects; the combined addition of PI15 and SFRP1 further promoted dentin formation capacity, confirming that PI15 can maintain the stemness of dental papilla cells in vivo, thereby promoting the dentin's ability to maintain proliferation and differentiation (the criteria for judging the repair effect are...). Figure 16 The smaller the remaining defect area in section B, the better the repair effect.

[0053] In the above experiment, the concentration ratios of each group were as follows: PI15 overexpression group (+PI15): 100 ng / mL; SFRP1 addition group (+SFRP1): 100 ng / mL; PI15 knockdown group (pLKO.1-TRC): Lentiviral MOI value 1-10; PI15 and SFRP1 combined supplement group (+PI15+SFRP1): each was administered at 50 ng / mL.

[0054] 2. Clarify the molecular mechanism by which PI15 regulates tooth germ epithelial-mesenchymal interaction and dentin formation: By integrating all experimental results, the core regulatory mechanism of PI15 was constructed, such as... Figure 17 As shown, PI15, specifically secreted by the dental epithelium, negatively inhibits the overactivation of the WNT pathway in the dental papilla mesenchyme by interacting with key molecules in the WNT pathway, thereby maintaining the differentiation of dental papilla mesenchymal cells into odontoblasts and ultimately regulating tooth germ development and dentin formation.

[0055] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A WNT signaling pathway inhibitor, characterized in that, The inhibitors include the PI15 protein or its encoding gene.

2. The inhibitor according to claim 1, characterized in that, The PI15 protein is a secretory protein derived from dental epithelium, and its amino acid sequence is shown in SEQ ID No.

1.

3. The inhibitor according to claim 2, characterized in that, The inhibitor also includes SFRP1 protein or its encoding gene; the SFRP1 protein is a secretory protein derived from dental papilla mesenchyme, and its amino acid sequence is shown in SEQ ID No.

2.

4. The use of the WNT signaling pathway inhibitor according to any one of claims 1-3 in the preparation of any one of the following products: 1) Products used to regulate mesenchymal cell morphogenesis and differentiation; 2) Products used to promote dentin formation or tooth tissue regeneration.

5. The application according to claim 4, characterized in that, The mesenchymal cells mentioned are dental papilla mesenchymal cells.

6. The application according to claim 5, characterized in that, The product used to regulate the morphogenesis and differentiation of mesenchymal cells achieves its regulatory effect by applying an effective dose of WNT signaling pathway inhibitor to mesenchymal cells, negatively regulating the expression of WNT pathway activation-related molecules Axin2, Ccnd1 and / or β-catenin in these cells, thereby inhibiting the differentiation of mesenchymal cells into odontoblasts or osteoblasts.

7. The application according to claim 5, characterized in that, The product used to regulate mesenchymal cell morphogenesis and differentiation works by knocking down or inhibiting the expression of PI15 protein or its encoding gene and SFRP1 protein or its encoding gene in the microenvironment of mesenchymal cells, thereby relieving the inhibition of the WNT signaling pathway in mesenchymal cells, promoting the expression of odontoblast differentiation markers Dlx5, Runx2 and / or MMP7 in mesenchymal cells, and thus promoting the differentiation of mesenchymal cells into odontoblasts.

8. The application according to claim 5, characterized in that, The product used to promote dentin formation or tooth tissue regeneration works by reducing the concentration or activity of PI15 / SFRP1 protein at the target site, thereby relieving the negative inhibition of the WNT pathway of dental papilla mesenchymal cells by PI15 / SFRP1, and thus promoting odontoblastic differentiation of dental papilla mesenchymal cells, achieving the function of promoting dentin formation or tooth tissue regeneration.

9. A pharmaceutical composition for promoting dentin formation or tooth tissue regeneration, characterized in that, The pharmaceutical composition comprises PI15 protein or recombinant PI15 protein, and / or SFRP1 protein or recombinant SFRP1 protein; the concentration ratio of PI15 protein to SFRP1 protein is selected as 1:1, 50 ng / mL each.

10. The pharmaceutical composition according to claim 9, characterized in that, The pharmaceutical composition is used to treat dentin defects, pulp damage, or tooth developmental abnormalities.

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