Use of axin1 in promoting odontoblastic differentiation of primary tooth dental pulp stem cells

Abstract

This invention discloses the application of AXIN1 in promoting odontoblastic differentiation of deciduous dental pulp stem cells. The study shows that with increasing odontoblastic differentiation induction time, the mRNA and protein expression of AXIN1 gradually increases; in a rat deep caries model, odontoblasts are active, and AXIN1 expression is significantly increased in reparative dentin; inhibition of AXIN1 expression leads to a significant decrease in the mRNA and protein levels of DSPP and DMP1, accompanied by decreased alkaline phosphatase activity and weakened mineralization capacity; while AXIN1 overexpression significantly increases the mRNA and protein expression levels of odontoblast-specific markers DSPP and DMP1, effectively enhances alkaline phosphatase activity, and significantly enhances cell mineralization capacity. This indicates that AXIN1 promotes odontoblastic differentiation of SHED cells.

Description

Technical Field

[0001] This invention relates to the field of odontogenic differentiation technology of deciduous tooth pulp stem cells, and more specifically, to the application of AXIN1 in promoting odontogenic differentiation of deciduous tooth pulp stem cells. Background Technology

[0002] Pulpitis and periapical periodontitis are two common oral diseases that cause irreversible damage to the dental pulp. Traditional clinical treatments, such as apical barrier surgery and root canal treatment, remove necrotic pulp and seal the root canal system with biological materials to control inflammation and promote the healing of periapical lesions. However, after treatment, the tooth loses its nutritional, immune, and sensory functions and cannot fully restore the original function of the pulp-dentin complex.

[0003] Primary dental pulp stem cells (SHEDs) originate from ectodermal neural crest tissue and possess multi-directional differentiation potential, capable of differentiating into odontoblasts, osteoblasts, adipocytes, nerve cells, vascular endothelial cells, hepatocytes, and pancreatic islet cells, showing broad application prospects in the field of tissue engineering. Investigating the molecular regulatory mechanisms and core factors of SHED differentiation into odontoblasts not only provides theoretical support for the construction of dental pulp-dentin complex bioengineering but also opens up innovative pathways for the clinical treatment of dental pulp-related diseases.

[0004] Axin1 is a key cytoskeletal protein regulating the formation of the embryonic developmental axis. Through interactions with signaling pathways such as Wnt, JNK, and TGF-β, it regulates cell proliferation, differentiation, apoptosis, and carcinogenesis, and participates in nervous system development processes such as neuronal migration and axon formation. Chinese patent CN119506203A discloses the application of AXIN1 in regulating osteogenic differentiation of deciduous tooth pulp stem cells (SHEDs), showing that overexpression of AXIN1 promotes osteogenic differentiation of SHEDs, while knockdown exhibits the opposite effect. That is, AXIN1 has a significant promoting effect on osteoblast differentiation and can effectively enhance bone formation efficiency. Because dentin formation and osteogenic differentiation differ significantly in cell fate determination, functional products, and regulatory mechanisms, there is currently no research on whether AXIN1 can regulate the differentiation of SHEDs into odontoblast-like cells. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of AXIN1 in promoting the dentin-to-dentinization differentiation of deciduous tooth pulp stem cells.

[0006] Another object of the present invention is to provide a method for promoting the dentin-to-dentinization differentiation of deciduous tooth pulp stem cells.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution: This invention first extracts and identifies primary SHED cells, and uses qRT-PCR and Western blotting to analyze the expression changes of AXIN1 during SHED differentiation toward odontoblasts. A rat model of deep caries in the maxillary first molar is constructed to observe changes in AXIN1 expression during reparative dentin formation. Small interfering RNA and lentiviral vector systems are used to inhibit and overexpress the AXIN1 gene. The gene expression levels of odontoblast-specific markers, dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1), are measured using qRT-PCR and Western blotting. Alkaline phosphatase (ALP) staining is used to assess the differentiation status of SHED cells, and Alizarin Red staining is used to analyze the mineralization capacity of SHED cells. The results showed that the mRNA and protein expression of AXIN1 gradually increased with the increase of odontoblast differentiation induction time. In the rat deep caries model, odontoblasts were active, and AXIN1 expression was significantly increased in reparative dentin. Inhibition of AXIN1 expression led to a significant decrease in the mRNA and protein levels of DSPP and DMP1, accompanied by a decrease in alkaline phosphatase activity and weakened mineralization capacity. In contrast, AXIN1 overexpression significantly increased the mRNA and protein expression levels of DSPP and DMP1, effectively enhanced alkaline phosphatase activity, and significantly improved mineralization capacity. This indicates that AXIN1 overexpression promotes odontoblast differentiation of SHED.

[0008] Therefore, the present invention first provides the application of AXIN1 in promoting the dentin-to-dentinization differentiation of deciduous tooth pulp stem cells.

[0009] This invention also provides the application of AXIN1 in the preparation of products that promote the dentin-to-dentinization differentiation of deciduous tooth pulp stem cells.

[0010] This invention also provides the use of formulations that promote AXIN1 overexpression in the preparation of products that promote dentin-to-dentinization differentiation of deciduous dental pulp stem cells.

[0011] Furthermore, the product upregulates the expression level of odontoblast-specific markers by overexpressing AXIN1, enhances alkaline phosphatase activity, and strengthens cell mineralization capacity, thereby promoting the odontoblastic differentiation of deciduous tooth pulp stem cells. This promotion of odontoblastic differentiation of deciduous tooth pulp stem cells refers to promoting their differentiation into odontoblast-like cells.

[0012] Preferably, the dentin-specific marker is selected from DSPP and / or DMP1.

[0013] The present invention also provides a method for promoting the odontoblastic differentiation of deciduous dental pulp stem cells, which involves treating deciduous dental pulp stem cells with an agent that promotes AXIN1 overexpression, followed by odontoblastic mineralization induction culture.

[0014] Furthermore, the formulation that promotes AXIN1 overexpression is an expression vector containing the AXIN1 gene.

[0015] Preferably, the expression vector is a lentiviral expression vector.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of AXIN1 in promoting odontoblastic differentiation of deciduous dental pulp stem cells. The study found that with increasing odontoblastic differentiation induction time, the mRNA and protein expression of AXIN1 gradually increased; in a rat deep caries model, odontoblasts were active, and AXIN1 expression was significantly increased in reparative dentin; inhibition of AXIN1 expression led to a significant decrease in the mRNA and protein levels of DSPP and DMP1, accompanied by decreased alkaline phosphatase activity and weakened mineralization capacity; while AXIN1 overexpression significantly increased the mRNA and protein expression levels of DSPP and DMP1, effectively enhanced alkaline phosphatase activity, and significantly improved cell mineralization capacity. This indicates that AXIN1 promotes odontoblastic differentiation of SHED. Attached Figure Description

[0017] Figure 1 Changes in AXIN1 mRNA expression during SHED odontoblast differentiation (*) P <0.05,** P <0.01, *** P <0.001, compared with the control group).

[0018] Figure 2 Changes in AXIN1 protein expression during SHED odontoblast differentiation (***) P <0.001, compared with the control group).

[0019] Figure 3 HE staining of deep caries in the second and first molars of rat control group (scale bar in the figure: top image 500 μm, bottom image 50 μm).

[0020] Figure 4 Immunohistochemical staining of AXIN1 in deep caries of the second and first molars of rats in the control group (scale bar in the figure: top image 500 μm, bottom image 50 μm, ***) P <0.001, compared with the control group).

[0021] Figure 5To interfere with AXIN1 protein expression (*) P <0.05,** P <0.01, *** P <0.001, compared with the control group).

[0022] Figure 6 To interfere with the changes in DSPP and DMP1 protein expression after AXIN1 (** P <0.01, compared with the control group).

[0023] Figure 7 To investigate the changes in ALP, DSPP, and DMP1 gene expression after interfering with AXIN1 (*) P <0.05,** P <0.01, compared with the control group).

[0024] Figure 8 ALP staining was performed to interfere with AXIN1 (scale bar in figure: 50 μm).

[0025] Figure 9 Alizarin Red staining and semi-quantitative analysis after interfering with AXIN1 (scale bar in figure: 50 μm, **) P <0.01, compared with the control group).

[0026] Figure 10 The results are for immunofluorescence (scale bar in the figure: 500 μm).

[0027] Figure 11 Gene expression after overexpression of AXIN1 (***) P <0.001, compared with the control group).

[0028] Figure 12 Protein expression after overexpression of AXIN1 (***) P <0.001, compared with the control group).

[0029] Figure 13 The expression levels of ALP, DMP1, and DSPP genes after overexpression of AXIN1 (**) P <0.01, *** P <0.001, compared with the control group).

[0030] Figure 14 The expression levels of DMP1 and DSPP proteins after overexpression of AXIN1 (**) P <0.01, compared with the control group).

[0031] Figure 15 ALP staining after AXIN1 overexpression (scale bar in figure: 50 μm).

[0032] Figure 16 Alizarin Red staining and semi-quantitative analysis after AXIN1 overexpression (scale bar in figure: 50 μm, ***) P <0.001, compared with the control group). Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0034] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0035] (1) α-MEM complete medium (α-MEM PM): Add 5 mL of fetal bovine serum (FBS) and 0.5 mL of penicillin / streptomycin to 44.5 mL of α-MEM medium, mix thoroughly, and seal and store at 4°C for later use.

[0036] (2) DMEM complete medium (DMEM PM): Add 5 mL of FBS and 0.5 mL of penicillin / streptomycin to 44.5 mL of high sugar DMEM medium, mix thoroughly, and seal and store at 4°C for later use.

[0037] (3) Odontoblast mineralization induction medium (OM): Add 5 mL of FBS, 0.5 mL of penicillin / streptomycin, 2.5 μL of 100 nM dexamethasone, 0.5 mL of 10 mmol / L sodium β-glycerophosphate, and 0.5 mL of 50 mg / L vitamin C to high-glucose DMEM medium, and bring the volume to 50 mL. Wrap the mixture in aluminum foil and store it in a light-proof environment at 4℃. Preparation of ex vivo deciduous tooth preservation solution: Add 1 mL of FBS and 0.5 mL of penicillin / streptomycin to 48.5 mL of α-MEM medium, mix thoroughly, and seal and store at 4℃ for later use.

[0038] AXIN1 axin 1 [Homo sapiens (human)], Gene ID: 8312, Transcript NCBI Reference Sequence: NM_003502.4.

[0039] Example 1: Effect of AXIN1 on odontoblast differentiation of human deciduous tooth pulp stem cells I. Experimental Methods 1. Isolation and culture of SHED This study was approved by the Medical Ethics Committee of the Affiliated Stomatological Hospital of Guangzhou Medical University (Ethics Approval No.: LCYJ2021025), and all study subjects and their legal guardians signed written informed consent documents. The experimental sample consisted of healthy children aged 6-8 years with no history of systemic or genetic diseases who visited the pediatric dental clinic of the hospital. The inclusion criteria were: primary teeth without pulp or periapical disease that needed to be extracted due to retention, and root resorption of less than 2 / 3.

[0040] Freshly extracted mandibular retained deciduous teeth were collected and preserved in ex vivo deciduous tooth preservation solution at 4°C. Primary cell (P0) extraction was completed within 4 hours. Before extraction, the tooth surface was washed twice with 75% ethanol and then soaked in 4% antibiotic-containing PBS for 1 min. The pulp was obtained from the root canal under sterile conditions and washed twice with a gradient of PBS containing 4% and 2% antibiotics. The pulp tissue was minced with a No. 11 blade, and 1 mL of 3 g / L type I collagenase and 1 mL of 4 g / L type II enzyme were added, respectively. The mixture was digested in a shaker at 37°C for 60 min until the pulp tissue became cloudy. An equal volume of α-MEM PM was added to stop the digestion. The mixture was centrifuged at 1000 rpm for 5 min, and the cell and tissue pellets were placed in a six-well plate. 1 mL of α-MEM PM containing 20% ​​FBS was added. After culturing for 3–7 days, the adhesion of the tissue pellets and the presence of cells emerging from the surrounding tissue were observed under a microscope. When the cells reached 80% coverage, they were passaged and cultured continuously. Human exfoliated deciduous tooth stem cells (SHED) from passages 3 to 6 (P3 to P6) were selected as materials for subsequent research.

[0041] 2. Flow cytometry detection After the P3 generation of SHED cells reached full confluence, they were digested with trypsin and then centrifuged at 250 rcf for 4 min. The cells were then washed twice with 6 mL PBS, followed by centrifugation after each wash. The cells were then resuspended in PBS containing 3% FBS to a final volume of 0.5 mL, and cell counting was performed. Approximately 5 × 10⁶ cells were injected into each light-protected Eppendorf tube. 6 Cells were collected and PBS containing 3% FBS was added to a total volume of 100 μL. Except for the control group, 5 μL of antibody (CD14, CD29, CD45, CD105, or CD146) was added to each EP tube. The cell suspension and antibody were gently mixed and incubated at room temperature in the dark for 30 min. The cell samples were then washed with PBS buffer, centrifuged at 800 rpm for 5 min, and the cell pellet was resuspended in 200 μL of PBS. Finally, flow cytometry was used to analyze and determine the percentage of cell surface antigen expression.

[0042] 3. SHED odontoblast differentiation induction culture SHED was induced to differentiate into odontoblasts, with the solution changed every 2 days.

[0043] 4. Construction of a deep caries model in SD rats A rat model of deep caries in the maxillary first molar was established to observe changes in AXN1 expression during reparative dentin formation. Eight SD rats, weighing 160-220 g, were used. They were anesthetized by intramuscular injection of Sutacetin 50 in the left hind limb (1 mL / g). Single-sided cavities were prepared on the mesial surface of both maxillary first molars. The cavity depth was approximately 1 mm; the cavities were not filled. The maxillary second and third molars and mandibular molars of each animal were left unfilled as blank controls. All cavity preparation was performed by one person, and the animals were sacrificed on postoperative day 30.

[0044] 5. Specimen preparation Immediately after euthanasia, the maxillae and mandibles of the experimental animals were separated and fixed in 4% paraformaldehyde fixative at 4°C for 48 hours. Subsequently, decalcification was performed for 4 weeks using 10% EDTA decalcification solution. After gradient dehydration with ethanol, clearing with xylene, and paraffin embedding, 4 μm thick sagittal serial sections were prepared, ensuring that each section completely included the maxillary first, second, and third molars. The sections were then processed, mounted on poly-L-lysine-coated slides, and stored at room temperature for later use.

[0045] 6. HE staining Three slides from each group of specimens were selected for HE staining, and after staining, the slides were mounted with resin and observed under a microscope.

[0046] 7. Immunohistochemical staining 1) Block endogenous peroxidase: Add 3% H2O2 to the tissue and incubate at room temperature for 15 min; 2) Wash the sections twice with PBS, 5 min each time; 3) Antigen retrieval: Tissue sections were placed in a solution of sodium citrate buffer at pH 6.0, heated in an autoclave for 15 minutes, and then allowed to cool naturally to room temperature. 4) Wash the sections twice with PBS, 5 minutes each time; 5) Antigen blocking: 5% BSA was added to the tissue and incubated at 37°C for 1 hour; 6) Incubation with primary antibody: Remove the blocking solution and add 100-400 µL of diluted primary antibody to each tissue section, incubate overnight at 4°C; 7) Wash the sections with PBS three times, 5 minutes each time; 8) Incubation of secondary antibody: Add diluted secondary antibody dropwise and incubate at 37°C for 1 h; 9) Wash the sections with PBS three times, 5 minutes each time; 10) DAB staining: Add freshly prepared DAB staining working solution and incubate at room temperature for 20 seconds; 11) Wash the sections with PBS to stop staining; 12) Hematoxylin counterstaining: stain with hematoxylin solution for 3.5 min, then rinse with running water for 10 min; 13) Dehydration and clearing: The gradient dehydration steps are as follows: 75% ethanol dehydration for 10 s; 95% ethanol dehydration for 10 s; anhydrous ethanol dehydration for 20 min; TO environmentally friendly clearing solution for 20 min; 14) Slide preparation: After the environmentally friendly transparent solution has dried, apply neutral resin to the edge of the tissue, cover with a coverslip, and scan the slide after it dries.

[0047] 8. Knockdown and overexpression of AXIN1 1) Knock-down of AXIN1 AXIN1 was knocked down using small interfering RNA transient transfection technology. Si-AXIN1 and its negative control Si-NC were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0048] The sense strand sequence of Si-AXIN1 is 5'-GCATCGTTGTGGCGTACTA-3' (SEQ ID No. 1); the antisense strand sequence is 5'-TAGTACGCCACAACGATGC-3' (SEQ ID No. 2).

[0049] siRNA negative control fragment: forward sequence: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID No. 3); reverse sequence: 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID No. 4).

[0050] In a 6-well plate, 2×10 5SHED cells were evenly seeded at a density of cells / well and transfected after 24 hours of complete cell adhesion. Si-AXIN1 and Si-NC were prepared to a working concentration of 20 μM using DEPC water. 2 μL of Lipofectamine 3000 was mixed with 500 μL of serum-free α-MEM medium and incubated at room temperature for 5 min to form transfection complex A. Separately, 40 pmol of siRNA was mixed with 500 μL of serum-free α-MEM medium and incubated at room temperature for 5 min to prepare transfection solution B. Complex A and transfection solution B were gently mixed and incubated at room temperature for 20 min to form the transfection complex system. After discarding the original culture medium, the cell monolayer was washed twice with PBS buffer, and the prepared transfection complex was added, followed by serum-free α-MEM medium to a final volume of 1 mL. Six hours after transfection, the medium was replaced with odontoblast mineralization induction medium (OM). This process was repeated 2-3 times as needed, and finally, an odontoblast differentiation capacity assay was performed.

[0051] 2) Overexpression of AXIN1 AXIN1 gene overexpression was performed using lentiviral stable transfection technology. The LV-AXIN1 lentiviral vector and the negative control LV-NC used in the experiment were both constructed by Guangzhou Dongze Biotechnology Co., Ltd. The specific procedure is as follows: SHED cells were transfected at a rate of 2 × 10⁻⁶ cells / year. 5 Inoculate each well with 2 mL of α-MEM complete medium (α-MEM PM) at a density of 1 virus / well. After 24 hours of incubation, perform virus transfection by adding 50 µL of LV-AXIN1 / LV-NC virus solution (titer 1×10⁻⁶) to each well. 8 A mixture of TU / mL and 950 µL serum-containing medium was used. After 24 h of transfection, the medium was replaced with α-MEM PM, and the cells were cultured for another 72 hours. Transfection efficiency was assessed using an inverted fluorescence microscope. Subsequently, cells were cultured in α-MEM PM containing 5 µg / mL puromycin to screen for SHED cells that stably passaged and overexpressed AXIN1. Subsequent experiments were conducted using odontoblast mineralization induction medium (OM) for 7–21 days.

[0052] 9. qRT-PCR detection of related gene expression 1) RNA extraction SHED cells induced to differentiate into odontoblasts after 7 days of inhibition or overexpression of the AXIN1 gene were used to extract total RNA using the Trizol reagent method.

[0053] 2) RT reaction Prepare the reverse transcription reaction solution (RT PrimerMix 2.0 μL + mRNA 6.0 μL + RNase-free dH2O 2.0 μL), mix well, and then use the AG reverse transcription program (37℃ for 15 min, 85℃ for 5 s, hold at 4℃). Dilute the cDNA sample to 100 μL / tube for the next experiment. Store in a refrigerator at 80°C.

[0054] 3) qRT-PCR The amount of cDNA reverse transcription product used for each sample was 2.0 μL. qRT-PCR was performed using SYBR Premix Ex Taq reagent on a qRT-PCR instrument (10 μL SYBR Premix Ex Taq, 1.6 μL primers, and an appropriate amount of sterile water). The reaction parameters were set as follows: 95℃ for 1 min; 95℃ for 30 s, 60℃ for 30 s, for a total of 40 cycles. GAPDH was selected as the reference gene, and 2... -ΔΔCт Expression levels were compared between experimental groups using a specific method. All primer sequences were prepared by Jereh Chemicals Co., Ltd. (China), and detailed primer information is shown in Table 1. Sequence homology was verified by BLAST.

[0055] Table 1 Sequence primers used for qRT-PCR

[0056] 10. Western blot detection of related protein expression 1) Protein extraction, concentration determination, and denaturation Protein extraction was performed on odontoblast-induced SHED, AXIN1-interferenced or overexpressed AXIN1-induced SHED after 14 days. After removing the culture medium, cells were washed three times with PBS, and 100 μL of RIPA protein lysis buffer containing 1 mM PMSF was added. Cells were shaken and pipetted for 30 minutes, then lysed on ice for 30 minutes. Cells were scraped layer by layer with a cell scraper and transferred to 1.5 mL centrifuge tubes. Centrifugation was performed at 12,000 rcf for 20 minutes at 4°C. The supernatant was collected into new 1.5 mL centrifuge tubes for quantitative analysis. Protein concentration was determined according to the BCA protein concentration assay kit instructions. 1 / 4 volume of SDS protein loading buffer was added to the sample and thoroughly mixed. The sample was denatured by heating in a 100°C incubator for 10 minutes and stored on ice for later use or for long-term storage. 80℃ refrigerator.

[0057] 2) Western Blot Select an appropriate gel concentration based on the molecular weight of the target protein, and prepare the separating gel and upper gel of the corresponding concentrations according to the instructions of the SDS-PAGE rapid gel preparation kit. Fix the prepared gels on the electrophoresis rack and place them in the electrophoresis tank. Add an appropriate amount of fresh electrophoresis buffer, and sequentially spot 5 μL of protein standard and 15 μg of protein sample into the sample wells. Electrophoresis is performed at 80 V for 30 min. After the markers are clearly separated, switch to 120 V for continuous electrophoresis until the bromophenol blue front reaches the bottom of the separating gel. Remove the gel plate and immerse it in transfer buffer, carefully separating the glass plate and the gel. Thoroughly wet the sponge pad, filter paper, and PVDF membrane (after methanol activation for 2 min), and assemble the transfer sandwich according to the structure of negative electrode plate-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-positive electrode plate. Correctly place it in the transfer tank, add transfer buffer, and transfer the membrane at 300 mA constant current for 90 min in an ice bath. Treat the PVDF membrane with the prepared rapid blocking solution for 30 min. According to experimental requirements, antibodies of specific concentrations were prepared using BSA (AXIN1 1:1000; DSPP 1:1000; DMP1 1:1000; GAPDH 1:50000). The blocked nitrocellulose membrane was placed in the antibody working solution and incubated overnight at 4°C. It was washed three times with TBST for 10 min each time, and then transferred to the fluorescent secondary antibody working solution (1:10000, diluted with 5% BSA) and reacted at room temperature in the dark for 1 h. It was washed three times with TBST for 10 min each time. After washing the membrane, the ultrasensitive chemical developing solution was prepared according to the instructions, mixed evenly, and dropped onto the surface of the PVDF membrane. It was then developed using a gel imaging system.

[0058] 11. ALP staining to detect cellular ALP activity levels LV-AXIN1, Si-AXIN1, and the control group SHED underwent 7 days of odontoblastic differentiation induction, and ALP staining was performed using the Beyotime BCIP / NBT alkaline phosphatase staining kit. The original culture medium was discarded, and the cells were washed three times with PBS in a shaker for 5 min each time. Cells were fixed with 4% paraformaldehyde for 15 min, and washed three times with PBS for 5 min each time. An appropriate amount of BCIP / NBT staining working solution was prepared according to the ratio of 10 μL BCIP solution (300X), 20 μL NBT solution (150X), and 3 mL alkaline phosphatase staining buffer. The prepared BCIP / NBT staining solution was added to completely cover the cells, and the cells were incubated at room temperature in the dark for 15 min. If the staining effect was consistent with expectations, the BCIP / NBT staining solution was removed, and the cells were rinsed twice with distilled water. The cells were then observed and photographed under a stereomicroscope.

[0059] 12. Alizarin Red Staining and Quantitative Analysis 1) Alizarin Red staining LV-AXIN1 and Si-AXIN1 control groups were induced to differentiate into odontoblasts by SHED for 21 days. The original culture medium was discarded, and the cells were washed three times with PBS in a shaker for 5 min each time. The cells were fixed with 4% paraformaldehyde for 15 min, washed three times with PBS for 5 min each time, and 150 μL of 0.5% alizarin red staining solution (pH 4.2) was added. The staining effect was observed regularly. If the staining effect was consistent with the expectation, the staining solution was removed, and the cells were washed 1-2 times with distilled water before observation and photography under a stereomicroscope.

[0060] 2) Semi-quantitative detection of CPC calcified nodules After the stained plate was air-dried, a 10% CPC solution was prepared, and 150 μL was injected into each well. The plate was then shaken on a shaker for 30 min to 1 h until the calcified crystals were completely dissolved. The solution was then transferred and diluted with a 10% CPC solution. The absorbance at 562 nm was measured using a microplate reader, with a 10% CPC solution used as a blank control.

[0061] 13. Statistical Analysis All experimental results in this study were validated through three independent replicates. All experimental data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism software. One-way ANOVA was used for statistical analysis among three or more groups, and Tukey's test was used for pairwise comparisons among multiple groups. The significance level was set at α = 0.05. P A value <0.05 indicates a statistically significant difference.

[0062] II. Results 1. Changes in AXIN1 expression during SHED odontoblast differentiation First, the changes in AXIN1 mRNA and protein expression levels during SHED differentiation into odontoblasts (1 d, 4 d, 7 d, and 14 d) were analyzed. The results showed that AXIN1 mRNA expression steadily increased with prolonged induction time. Figure 1 The expression of AXIN1 protein changed in a consistent manner, reaching its peak level 14 days after odontoblastic differentiation induction. Figure 2 ).

[0063] 2. Changes in AXIN1 expression during reparative dentin and odontoblast formation. A rat model of deep caries in the maxillary first molar was subjected to HE and AXIN1 IHC. Results showed that after 30 days, reparative dentin significantly increased, and the pulp cavity narrowed. In high-power fields, the reparative dentin exhibited tubular structures, but these were less pronounced than in normal dentin. Newly differentiated odontoblast-like cells were arranged in a significantly more regular pattern. Figure 3Secondly, AXIN1 expression is increased during reparative dentin formation. Figure 4 ).

[0064] 3. Effect of knockdown of AXIN1 on the odontoblast differentiation ability of SHED The endogenous expression of AXIN1 in SHED cells was inhibited using siRNA transfection technology, and the gene inhibition effect was detected by qRT-PCR and Western blot. The results showed that both the mRNA expression level and protein synthesis of AXIN1 were significantly reduced in the SI-AXIN1 group. Figure 5 This indicates that the AXIN1 gene was effectively silenced. In the analysis of odontoblast differentiation markers, compared to the SI-NC control group, the SI-AXIN1 experimental group showed a significant decrease in the mRNA synthesis of alkaline phosphatase, dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP). Figure 6 Meanwhile, the protein expression levels of DMP1 and DSPP were also significantly downregulated. Figure 7 The differences between the two groups were statistically significant. Further analysis using ALP and ARS mineralization staining showed that the AXIN1 silencing group had significantly reduced cellular ALP activity and mineralized nodule formation capacity. Figure 8 , Figure 9 This provides data support for the role of AXIN1 in the regulatory mechanism of SHED cell differentiation.

[0065] 4. Effect of AXIN1 overexpression on the odontoblast differentiation ability of SHED An AXIN1 overexpression model was constructed using lentiviral stable transfection technology. The overexpression efficiency was verified by inverted fluorescence microscopy, qRT-PCR, and Western blotting analysis. Experimental data showed that SHED cells treated with LV-AXIN1 exhibited significant green fluorescence under a fluorescence microscope. Figure 10 Furthermore, the expression levels of both AXIN1 mRNA and protein were significantly upregulated. Figure 11 , Figure 12 This confirmed the successful construction of the AXIN1 gene overexpression model. Further analysis of odontogenic differentiation markers showed that, compared to the LV-NC control group, the mRNA expression of ALP, DMP1, and DSPP was significantly enhanced in the LV-AXIN1 experimental group. Figure 13 Meanwhile, the expression levels of DMP1 and DSPP proteins also showed a statistically significant increase. Figure 14 In functional validation experiments, ALP staining and Alizarin Red staining results showed that the alkaline phosphatase activity of cells overexpressing AXIN1 was significantly enhanced, and more obvious mineralized nodule formation was observed. Figure 15 , Figure 16This study confirmed that AXIN1 overexpression can effectively promote odontoblast differentiation and mineralization in SHED.

[0066] In summary, this invention is the first to discover that AXIN1 is activated during the differentiation of SHED into odontoblast-like cells. By interfering with AXIN1 expression, the differentiation of SHED into odontoblast-like cells was inhibited; conversely, overexpression of AXIN1 promoted the differentiation of SHED into odontoblast-like cells. This invention reveals for the first time the core regulatory role of AXIN1 in promoting the differentiation of SHED into odontoblast-like cells.

Claims

1. Application of AXIN1 in promoting the dentin-to-dentinization differentiation of deciduous tooth pulp stem cells.

2. Application of AXIN1 in the preparation of products that promote dentin differentiation of deciduous tooth pulp stem cells.

3. Application of formulations that promote AXIN1 overexpression in the preparation of products that promote dentin-to-dentinization differentiation of deciduous tooth pulp stem cells.

4. The application according to claim 2 or 3, characterized in that, The product upregulates the expression level of dentin-specific markers by overexpressing AXIN1, enhances alkaline phosphatase activity, and improves cell mineralization capacity, thereby promoting the dentin-to-dentin differentiation of deciduous tooth pulp stem cells.

5. The application according to claim 4, characterized in that, The dentin-specific markers are selected from dentin salivary phosphoprotein and / or dentin matrix protein 1.

6. The application according to claim 3, characterized in that, The formulation that promotes AXIN1 overexpression is an expression vector containing the AXIN1 gene.

7. The application according to claim 6, characterized in that, The expression vector is a lentiviral expression vector.

8. A method for promoting the dentin-to-dentinization differentiation of deciduous tooth pulp stem cells, characterized in that, Primary dental pulp stem cells were treated with an agent that promotes AXIN1 overexpression, followed by odontoblast mineralization induction culture.

9. The method according to claim 8, characterized in that, The formulation that promotes AXIN1 overexpression is an expression vector containing the AXIN1 gene.

10. The method according to claim 9, characterized in that, The expression vector is a lentiviral expression vector.

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