Application of Axin1 in regulation and control of osteogenic differentiation of dental pulp stem cells of deciduous teeth

Abstract

The invention discloses application of Axin1 in regulation and control of osteogenic differentiation of dental pulp stem cells of deciduous teeth. The research shows that the overexpression and knock-down of Axin1 do not influence the proliferation of SHED and the characteristics of MSC. However, overexpression of Axin1 promotes migration of SHED, and this effect can be weakened by Axin1 silencing. Overexpression of Axin1 promotes osteogenic differentiation of SHED, and knock-down of Axin1 shows a reverse effect. In mechanism, overexpression of Axin1 obviously improves the expression level of phosphorylated ERK1 / 2, so that osteogenic differentiation of SHED is promoted. The promotion effect of overexpression of Axin1 on osteogenic differentiation of SHED can be eliminated by using an ERK1 / 2 chemical inhibitor for treatment, which indicates that Axin1 plays a role as a key regulatory factor for osteogenic differentiation in SHED through an ERK1 / 2 pathway. A research result shows that Axin1 can be used as a potential new target and is used for treating diseases related to bone regeneration by applying SHED.

Description

Technical Field

[0001] The present invention relates to the technical field of osteogenic differentiation of mesenchymal stem cells, and more specifically, to the application of Axin1 in regulating osteogenic differentiation of deciduous tooth pulp stem cells. Background Art

[0002] Regenerative medicine has great potential for patients with bone problems, which have a great negative impact on patients' quality of life and are a major medical burden. Therefore, bone repair and regeneration are one of the main goals of bone tissue engineering. Traditional autologous and allogeneic bone transplantation has many limitations, such as limited bone resources, low osteoinduction and osteointegration capabilities, and immune rejection of allogeneic transplants. In recent years, the method of regenerative therapy using tissue engineered bone (TEB) composed of mesenchymal stem cells (MSCs) + scaffold materials + cytokines has been widely studied. This method can greatly improve the safety and effectiveness of bone regeneration therapy while avoiding invasive surgery and other processes. It is now considered to be one of the best strategies for treating large bone defects.

[0003] As an important type of MSCs, stem cells from human exfoliated deciduous teeth (SHED) derived from the pulp tissue of human exfoliated deciduous teeth are an ideal source for cell therapy due to their rich tissue and easy access. SHED have low immunogenicity and few ethical issues. In addition, they have high stemness and can differentiate into multiple cell lineages such as osteoblasts, chondrocytes, odontoblasts, adipocytes and neurons. SHED has strong angiogenic ability and can release high levels of cytokines, such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). It has been previously reported that the secretion ability of cytokines can affect the osteogenic differentiation of MSCs. In addition, compared with dental pulp stem cells (DPSCs) and bone marrow mesenchymal stem cells (BMSCs), SHED has higher proliferation and osteogenic activity because it produces higher levels of osteocalcin (OCN) and alkaline phosphatase (ALP), and expresses more basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP2) after differentiation. Many preclinical studies have confirmed that SHED is particularly suitable for bone regeneration.

[0004] Axin is a cell-structured protein that regulates the formation of the body axis during embryonic development. It is associated with multiple signaling pathways such as Wnt, JNK, TGF-β, and AMP-activated protein kinase (AMPK), thereby affecting biological processes such as cell proliferation, differentiation, apoptosis, and carcinogenesis. It is an important gene that controls processes such as cell proliferation, differentiation, and cell cycle. It has been confirmed that the Axin family has two members: Axin1 and its homolog Axin2 (Axil). They have the same structural domain and functional consistency. Axin1 is widely expressed in tissues, while Axin2 is only expressed in specific tissues and developmental stages.

[0005] Recently, the research on Axin in bone tissue has gradually attracted the attention of scholars. It has been reported that Axin1 knockdown can inhibit osteoblast apoptosis induced by Porphyromonas gingivalis lipopolysaccharide. In addition, inhibition of Axin1 in osteoblast precursor cells was found to lead to postnatal bone growth defects due to inhibition of osteoclast formation. In addition, it has been shown that deletion of Axin1 in condylar chondrocytes leads to condylar cartilage degeneration in mice by activating β-catenin and ERK1 / 2 signaling pathways. However, there is a lack of relevant studies on the effect of Axin1 on the function of MSCs, especially the functional role, expression level and molecular regulation mechanism of MSCs in the osteogenic differentiation process. In particular, the effect of Axin1 on the biological characteristics and osteogenic differentiation potential of SHED has not been reported. Summary of the invention

[0006] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide the application of Axin1 in the osteogenic differentiation of dental mesenchymal stem cells. The present invention shows that overexpression and knockdown of Axin1 do not affect the proliferation and MSC characteristics of SHED. However, overexpression of Axin1 promotes the migration of SHED, and this effect can be weakened by silencing Axin1. Overexpression of Axin1 promotes the osteogenic differentiation of SHED, while its knockdown shows the opposite effect. It shows that Axin1 can be used as a potential new target for the application of SHED to treat diseases involving bone regeneration.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions: This study aims to investigate the effect of Axin1 expression on the characteristics of MSCs (SHED) cells, especially their osteogenic differentiation potential. By studying SHED, we attempt to further understand how changes in Axin1 expression affect the osteogenic differentiation of MSCs and determine whether Axin1 can be used as a potential new target for the application of SHED to treat diseases involving bone regeneration.

[0008] Specifically, the present invention isolates SHED and uses a lentivirus containing the Axin1 gene or a small interfering RNA targeting Axin1 to regulate the expression of Axin1. The present invention first studies the effects of Axin1 expression on the proliferation, migration and mesenchymal characteristics of SHED by CCK8 method, Transwell experiment and flow cytometry (FCM), respectively. In addition, the present invention also performs alkaline phosphatase (ALP) activity detection, Alizarin red staining, Western blotting (WB), real-time quantitative polymerase chain reaction (qRT-PCR) and in vivo osteogenesis experiments to determine the effect of Axin1 expression on SHE osteogenesis. The present invention finds that overexpression and knockdown of Axin1 do not affect the proliferation and MSC characteristics of SHED. However, overexpression of Axin1 promotes the migration of SHED, and this effect can be weakened by Axin1 silencing. Overexpression of Axin1 promotes the osteogenic differentiation of SHED, while its knockdown shows the opposite effect. Mechanistically, overexpression of Axin1 significantly increases the expression level of phosphorylated ERK1 / 2, thereby promoting the osteogenic differentiation of SHED. Treatment with chemical inhibitors of ERK1 / 2 abolished the promoting effect of Axin1 overexpression on osteogenic differentiation of SHED, indicating that Axin1 functions as a key regulator of osteogenic differentiation in SHED through the ERK1 / 2 pathway.

[0009] Therefore, the present invention provides the use of Axin1 in positively regulating the osteogenic differentiation of dental mesenchymal stem cells.

[0010] The present invention also provides the use of Axin1 as a target in the preparation of a product that promotes the osteogenic differentiation of dental mesenchymal stem cells.

[0011] The present invention also provides the use of a preparation for promoting overexpression of Axin1 in preparing a product for promoting osteogenic differentiation of dental mesenchymal stem cells.

[0012] Furthermore, the product increases the expression level of osteogenesis-related genes by overexpressing the Axin1 gene, thereby promoting the osteogenic differentiation of dental mesenchymal stem cells.

[0013] Furthermore, the osteogenesis-related genes include ALP, RUNX2, COL1 and OCN.

[0014] Furthermore, the product promotes the osteogenic differentiation of dental mesenchymal stem cells by overexpressing the Axin1 gene and increasing the expression level of phosphorylated ERK1 / 2.

[0015] Furthermore, the product is a drug for treating bone loss.

[0016] The present invention also provides a method for promoting osteogenic differentiation of dental mesenchymal stem cells, which comprises first treating the dental mesenchymal stem cells with a preparation promoting overexpression of Axin1, and then culturing the treated dental mesenchymal stem cells for osteogenic differentiation induction.

[0017] Furthermore, the preparation for promoting overexpression of Axin1 is an expression vector containing the Axin1 gene.

[0018] Preferably, the vector is a lentiviral vector.

[0019] Furthermore, the osteogenic differentiation induction culture is to culture the treated dental mesenchymal stem cells in an osteogenic differentiation induction medium.

[0020] As a preferred embodiment, the method for promoting the osteogenic differentiation ability of dental mesenchymal stem cells comprises the following steps: S1. Obtain dental mesenchymal stem cells and perform subculture; S2. Construction of an expression vector for overexpressing the Axin1 gene; S3. The constructed expression vector for overexpressing the Axin1 gene is transfected into dental mesenchymal stem cells, and then the transfected dental mesenchymal stem cells are induced and cultured in an osteogenic differentiation induction medium.

[0021] Preferably, the osteogenic differentiation induction medium is prepared by adding 10% fetal bovine serum, 100 mmol / L dexamethasone, 0.05 mmol / L vitamin C and 10 mmol / L glycerol phosphate to DMEM.

[0022] Furthermore, the dental mesenchymal stem cells include but are not limited to deciduous dental pulp stem cells isolated from deciduous teeth.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention explores the effect of Axin1 expression on the cell characteristics of MSCs (SHED), especially its osteogenic differentiation potential. The present study showed that overexpression and knockdown of Axin1 did not affect the proliferation and MSC characteristics of SHED. However, overexpression of Axin1 promoted the migration of SHED, and this effect could be weakened by Axin1 silencing. Overexpression of Axin1 promoted the osteogenic differentiation of SHED, while its knockdown showed the opposite effect. Mechanistically, overexpression of Axin1 significantly increased the expression level of phosphorylated ERK1 / 2, thereby promoting the osteogenic differentiation of SHED. Treatment with chemical inhibitors of ERK1 / 2 eliminated the promoting effect of Axin1 overexpression on the osteogenic differentiation of SHED, indicating that Axin1 plays a role as a key regulator of osteogenic differentiation in SHED through the ERK1 / 2 pathway. The results of the present study indicate that Axin1 can be used as a potential new target for the treatment of diseases involving bone regeneration using SHED. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results show the effect of Axin1 expression on SHED proliferation and migration. Figure 1 A in the figure shows the expression of Axin1 mRNA after SHED was transfected or transduced with siRNA targeting Axin1 or lentiviral particles overexpressing Axin1; B shows the expression of Axin1 protein in SHED after SHED was transfected with siRNA targeting Axin1 or lentiviral particles overexpressing Axin1, and the histogram shows the quantitative results of band intensity; C shows the results of inverted fluorescence microscopy photography two days after SHED was transfected with lentivirus overexpressing Axin1; D shows the effect of Axin1 silencing or overexpression on SHED migration, and E shows the effect of Axin1 silencing (left) or overexpression (right) on SHED proliferation; Si-Axin1, siRNA targeting Axin1; Si-NC, non-targeting control siRNA; LV-Axin1, lentiviral particles targeting Axin1; LV-NC, non-targeting control lentiviral particles. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0025] Figure 2 The results show the effect of Axin1 overexpression on the expression of surface marker antigens of SHED cells. Figure 2 A is the flow cytometry result of SHED cell surface marker antigens after SHED was transduced with negative control lentiviral particles (LV-NC); B is the flow cytometry result of SHED cell surface marker antigens after SHED was transduced with lentiviral particles containing Axin1 transgene (LV-Axin1) to overexpress Axin1.

[0026] Figure 3 The results show the effect of Axin1 expression on SHED osteogenesis in vitro. Figure 3 A in the middle is the result of RT-PCR detection of osteogenic genes after osteogenic induction in SHED culture for 7 days; B is the result of protein immunoblotting of osteogenic genes after osteogenic induction in SHED culture for 7 days, and the histograms (middle and right) show the quantitative results of band intensity; C is the result of the effect of Axin1 expression on ALP activity in SHED, and the histogram is the quantitative result of ALP; D is the result of the effect of Axin1 expression on matrix mineralization in SHED, and the histogram is the quantitative result of Alizarin Red staining by spectrophotometry; NC, negative control siRNA; Si-Axin1, siRNA targeting Axin1; LV-NC, non-targeting control lentiviral particles; LV-Axin1, lentiviral particles containing Axin1 transgene; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0027] Figure 4 The results show the effect of Axin1 expression on SHED osteogenesis in vivo. Figure 4 A is a reconstructed three-dimensional micro-computed tomography (micro-CT) image of a representative tissue-engineered bone construct of an implant loaded with SHED transduced with LV-Axin1 or LV-NC (left), and the ratio of the new bone volume (BV) to the original existing tissue volume (TV) (BV / TV) of the bone construct of an implant loaded with SHED transduced with LV-Axin1 and LV-NC (right, n=3 in each group), data are expressed as mean±SD, *P<0.05; B is the results of hematoxylin-eosin (H&E) staining and osteocalcin Masson staining of implants with different Axin1 expressions.

[0028] Figure 5 The results of RNA sequencing analysis of SHED cells overexpressing Axin1 and control cells. Figure 5 A is the hierarchical clustering result of differentially expressed mRNAs between Axin1-overexpressed SHED and control cells; B is the volcano plot of differentially expressed mRNAs between Axin1-overexpressed SHED and control SHED; C is the functional classification result of differentially expressed mRNAs induced by Axin1 overexpression in the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis; D is the sequencing result; E is the Peptrack proteomics database result.

[0029] Figure 6 This is the functional validation result of Axin1 activating ERK1 / 2 pathway to promote osteogenic differentiation of SHED. Figure 6A shows the effect of Axin1 knockdown on ERK phosphorylation; B shows the effect of Axin1 overexpression on ERK phosphorylation, left: protein immunoblot images, right: semi-quantitative results of p-ERK / ERK ratio in SHED cells with or without Axin1 overexpression and control cells; C shows the effect of ERK inhibitor FR180204 on ERK phosphorylation in SHED cells with and without Axin1 overexpression, left: protein immunoblot images, right: quantitative analysis of p-ERK / ERK ratio; D shows the effect of ERK inhibition on matrix mineralization in SHED cells with and without Axin1 overexpression, left: Alizarin red staining images of calcified nodules, right: histogram showing the quantitative results of Alizarin red staining by spectrophotometry, values ​​are expressed as mean ± standard deviation. NC: negative control siRNA; Si-Axin1: siRNA targeting Axin1; LV-NC, non-targeting control lentiviral particles; LV-Axin1, lentiviral particles containing Axin1 transgene. ERK: extracellular signal-regulated kinase. *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0031] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0032] Example 1 Effects of Axin1 on the biological properties of deciduous tooth pulp stem cells 1. Experimental Methods 1. Isolation and cultivation of SHED SHED were isolated from retained deciduous teeth of healthy children aged 6-12 years. This study was approved by the Medical Ethics Committee of the Affiliated Stomatological Hospital of Guangzhou Medical University (approval number: KY2019008), and informed written consent was obtained from the patients' parents. Pulp tissue was isolated and minced under sterile conditions, digested with 3 g / L collagenase I (Gibco, USA) and 4 g / L dispase for 30 min, centrifuged for 5 min, and the collected cells were inoculated in T25 culture flasks with DMEM medium (Gibco, USA) containing 0% fetal bovine serum (Gibco, USA) and 1% penicillin / streptomycin (Gibco, USA). When the cells reached 80% confluence, they were trypsinized and continued to be subcultured. As described below, SHED was determined as a positive and negative surface marker of MSCs (CD73 + / CD90 + / CD105 + / CD34 − / CD45 − ) and their osteogenic differentiation ability (osteoinduction). SHEDs of passages 3-6 were collected for subsequent experiments.

[0033] 2. Overexpression and knockdown of Axin1 in SHED SHED were transduced with recombinant lentivirus containing human Axin1 gene to overexpress Axin1, or transfected with Axin1 siRNA to knockdown Axin1 expression. For overexpression, Axin1-GFP lentiviral particles (LV-Axin1) and negative control empty vector-GFP control particles (LV-NC) (Shanghai GeneCare, China) were used and treated at the indicated multiplicity of infection in serum-free medium. After incubation with virus for 6 h, fresh complete medium was replaced. Transduction efficiency was checked under a fluorescence microscope 48 h later, and Axin1 expression was determined by real-time polymerase chain reaction (RT-PCR). For knockdown of Axin1, SHED were seeded in 6-well plates, and siRNA was transfected using Lipofectamine 3000 (Invitrogen, USA) when the cells reached 75%-90% confluence. Pre-designed and validated siRNA targeting Axin1 (Si-Axin1, target sequence: GCATCGTTGTGGCGTACTA) and control non-targeting siRNA (Si-NC, F: 5`-UUCUCCGAACGUGUCACGUTT-3`, R: 5`-ACGUGACACGUUCGGAGAATT-3`) were purchased from Ruibo Bio (Guangzhou, China). After incubation for 72 h, RT-PCR was performed to detect the expression of Axin1.

[0034] 3. Detection of cell surface markers by flow cytometry Surface antigens (CD73, CD19, CD105, CD90, CD45, CD34) of SHED were analyzed by flow cytometry (FCM) using a flow cytometry kit (BD Pharmingen, USA). CD73, CD19, and CD105 were labeled with phycoerythrin (PE). CD90 and CD45 were labeled with phycoerythrin-CY5 conjugate (PE-CY5). CD34 was labeled with fluorescein isothiocyanate (FITC) for cells treated with Axin1 siRNA or si-control, and with PE for cells treated with Axin1-GFP lentiviral particles or LV-NC.

[0035] 4. CCK8 detection of cell proliferation Cell proliferation was assessed using the CCK8 kit (Dojindo, Japan) following the manufacturer's instructions. SHED was plated at 1 × 103 Cells were seeded in 96-well plates at a density of 10 cells / mL. Cell proliferation was measured 1, 3, 5, and 7 days after seeding. After incubation with CCK8 reagent for 2 hours, the absorbance was read at 450 nm on a microplate reader (Thermo Fisher, USA). The experiment was repeated three times.

[0036] 5. Transwell assay for cell migration 5×10 SHED cells were transfected into each well. 4 The density of cells was seeded in the upper chamber of the Transwell. The cells grown in the upper chamber were cultured with serum-free medium, while complete medium was added to the lower chamber of the Transwell. After incubation for 48 h, the non-migrated cells on the filter membrane were carefully wiped off with a cotton swab. The migrated cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet (Shanghai Bogu, China). Five random fields of view of crystal violet-positive cells were photographed under an inverted microscope (Leica Instruments, USA). The cell density was analyzed using ImageJ software.

[0037] 6. RT-PCR detection of Axin1 gene expression level SHED was added at 1 × 10 5 The cells were seeded in 6-well culture plates at a density of 100 cells / mL. Total RNA was extracted from the cultured cells using TRIzol reagent (Invitrogen, USA). mRNA (500 ng) was reverse transcribed into cDNA using PrimeScript RT Master Mix Kit (Takara, Japan). RT-PCR was performed on cDNA using SYBR® Premix Ex Taq TM (Takara, Japan). The primer sequences for RT-PCR are shown in Table 1. The ABI Step-OnePlus Real-Time PCR System was used for 30 seconds at 95°C, followed by 40 cycles of 5 seconds at 95°C and 30 seconds at 60°C. System: SYBR® Premix Ex Taq TM 10μL, PCR Forward Primer (10μM) 0.4μL, PCR Reverse Primer (10μM) 0.4μL, ROX Reference Dye (50×) 0.4μL, cDNA 2.0μL, RNase Free Water 6.8μL, total volume 20μL. GAPDH was used as an internal reference, and the 2-ΔΔCt method was used to calculate the relative expression of the target gene.

[0038] Table 1 Sense and antisense primers for real-time reverse transcription polymerase chain reaction (RT-PCR)

[0039] 7. Statistical analysis The experiments were carried out in parallel for 3 times, and the experimental data were expressed as mean ± standard deviation. SPSS 20.0 statistical software was used for T test analysis, and GraphPad Prism was used for drawing. P < 0.05 The difference was statistically significant.

[0040] 2. Results In order to investigate the function of Axin1 in SHED, the present invention used siRNA targeting Axin1 and lentiviral particles (LV-Axin1) containing Axin1 cDNA sequence to regulate the expression of Axin1. To silence or overexpress Axin1, SHED was transfected with small interfering RNA (siRNA) targeting Axin1 or transduced with lentiviral particles (LV), respectively, and the expression of Axin1 mRNA in SHED was evaluated by quantitative reverse transcription polymerase chain reaction (RT-PCR). Three siRNAs targeting Axin1 (Si-Axin1-1, 2, and 3) were tested, and Si-Axin1-1 was selected for subsequent experiments, as shown in Figure 2 . Figure 1 Middle A and Figure 1 As shown in Figure B, after 3 days of treatment, the expression of Axin1 was successfully silenced by siRNA transfection and enhanced by lentiviral transduction. Using the GFP transgene in the lentiviral construct, the present invention tracked the lentiviral-mediated gene overexpression. As early as 2 days after lentiviral transduction, the fluorescent protein was highly expressed in SHED ( Figure 1 Axin1 knockdown and overexpression were achieved continuously during the experiment ( Figure 3 Axin1 is a scaffold protein, and the present invention further analyzed the effect of its expression on SHED migration using Transwell assay. Interestingly, Axin1 silencing significantly reduced SHED migration compared with cells treated with non-targeting control siRNA (Si-NC) ( Figure 1 In contrast, overexpression of Axin1 via lentiviral transduction resulted in a significant increase in SHED migration ( Figure 1 Middle D). Through cell viability experiments, it was found that Axin1 silencing or overexpression had no effect on the proliferation of SHED ( Figure 1Figure 5 (E). SHEDs belong to MSCs. To characterize SHEDs as MSCs, CD73, CD90, and CD105 were selected as positive cell surface markers of MSCs, while CD19, CD34, and CD45 were selected as negative markers. As expected, more than 99% of SHEDs expressed CD73, CD90, and CD105, while CD19, CD34, and CD45 were barely detectable (less than 1% of SHEDs). Notably, overexpression of Axin1 by lentiviral transduction had no effect on the expression of these MSC surface biomarkers, suggesting that increased Axin1 expression does not affect the stemness of SHEDs ( Figure 2 Middle A and Figure 2 Middle B).

[0041] In summary, interference and overexpression of Axin1 did not affect the stemness and proliferation ability of SHED, and SHED still had the characteristics of mesenchymal stem cells. The migration ability of SHED was significantly reduced after interference with Axin1, and the migration ability of SHED was significantly enhanced after overexpression of Axin1, which indicated that positive regulation of Axin1 could promote SHED migration.

[0042] Example 2 Effect of Axin1 expression on osteogenic differentiation of SHED 1. Experimental Methods 1. Osteogenic differentiation Osteogenic induction medium was prepared by adding 10% fetal bovine serum, 100 mmol / L dexamethasone, 0.05 mmol / L vitamin C, and 10 mmol / L glycerol phosphate (all purchased from Shanghai Sangon Biotechnology Co., Ltd.) to 90 mL of DMEM and stored at 4°C. During in vitro osteogenic differentiation, SHED cells with Axin1 knockdown or overexpression were seeded in 6-well plates and induced with osteogenic induction medium, with the medium replaced every 2-3 days.

[0043] 2. RT-PCR detection of SHED osteogenic gene mRNA expression level The experimental method of Example 1 was performed in step 6 to detect the gene expressions of runt-related transcription factor 2 (Runx2), type I collagen (COL1), alkaline phosphatase (ALP) and osteocalcin (OCN).

[0044] 3. Western Blot Analysis The transfected SHED was treated with RIPA buffer (Thermo Fisher, USA) and centrifuged at 12,000 rpm for 15 min at 4°C to collect the protein supernatant. The total protein concentration of each sample was measured using a BCA kit (Thermo Fisher, USA). The protein samples (20 μg) were separated by 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with 5% skim milk in Tris-buffered saline (TBST) containing 0.1% Tween-20 at 37°C for 2 h and then incubated with different primary antibodies overnight at 4°C: rabbit anti-human GAPDH (CellSignaling Technology, USA), rabbit anti-human Axin1 (Cell Signaling Technology, USA), rabbit anti-human ALP (Cell Signaling Technology, USA), and rabbit anti-human COL1 (Cell Signaling Technology, USA). After washing with TBST for 10 min, the membrane was incubated with HRP-conjugated affinity-purified goat anti-rabbit IgG for 2 h at room temperature. Protein bands were visualized using a Western Blot Imaging System (Bio-Rad, USA).

[0045] 4. Alkaline phosphatase activity detection 1×10 SHED cells were transfected into each well. 4 Cells were seeded in 48-well plates at a density of 10 cells per well for osteogenic induction. ALP activity was detected at 405 nm using an ALP activity detection kit (Nanjing Jiancheng, China). The total protein content of each sample was determined using a BCA kit (ThermoFisher, USA). The values ​​of ALP activity relative to the control group were normalized to the total protein content. For ALP staining, transfected SHED were washed with PBS and fixed with 4% paraformaldehyde for 30 min. After washing with PBS three times, the cells were stained using an NBT / BCIP staining kit (Biyuntian, China) according to the manufacturer's instructions. Images were taken under a microscope (Leica Instruments, USA).

[0046] 5. Alizarin red staining and quantification 2×10 SHED cells were transfected into each well. 4Cells were seeded in 48-well plates at a density of 10 cells / mL. After 14 days of culture, cells were fixed with 95% ethanol for 30 min at room temperature (RT), rinsed three times with deionized water, stained with 5% Alizarin Red (pH = 4.2, SigmaAldrich, USA) for 5–10 min, washed repeatedly with deionized water, and then dried at RT. The dried plates were placed under a stereomicroscope (Leica Instruments, USA) for photography. For quantification of calcified nodules, Alizarin Red dissolved in 10% cetylpyridinium chloride (CPC, Beyotime, China) was added to the plates. After incubation for 1 h at RT, the absorbance was read at 562 nm on a microplate reader (Thermo Fisher, USA). The final calcium concentration was normalized to the total protein concentration.

[0047] 6. Bone formation experiment and micro-computed tomography (Micro-CT) analysis Axin1-overexpressing SHED and control groups were induced with osteogenic medium for 1 week and then harvested for in vivo studies. SHED incubated with Bio-Oss Collagen (Geistlich, Germany) scaffolds at 37°C for 2 h were implanted subcutaneously on the backs of BALB / c homozygous nude mice (5 weeks old, three mice per group). After 8 weeks, the implants were removed and fixed with 4% paraformaldehyde. The animal experimental procedures were approved by the Animal Experiment Ethics Committee of Guangdong Quality Supervision Medical and Health Device Inspection Station (approval number: 2021012001). Micro-CT analysis was performed using a high-resolution Inveon Micro-CT (Bruker, Germany) with an X-ray source of 71 kV, a light tube current of 100 μA, and a scanning accuracy of 10 μm. Image slices were reconstructed using Micro-CT image analysis software (InveonResearch Workplace). The ratio of new bone volume to existing tissue volume (BV / TV) was calculated.

[0048] 7. Histological and Histomorphometric Analysis The tissues were decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 4 weeks, dehydrated and embedded in paraffin. The tissues were sectioned and stained with hematoxylin and eosin (H&E) or Masson's trichrome staining. Ten random fields of view of each section were photographed under a microscope.

[0049] 8. Statistical analysis The experiments were carried out in parallel for 3 times, and all data were expressed as mean ± standard deviation. SPSS 20.0 statistical software was used for T test analysis and One-Way ANOVA analysis, and GraphPad Prism was used for drawing. P < 0.05 The difference was statistically significant.

[0050] 2. Results SHEDs transfected or transduced with different levels of Axin1 expression were cultured in osteogenic induction medium. The present invention first detected the expression of several osteogenesis-related genes, including ALP, RUNX2, COL1, and OCN, after 7 days of osteogenic induction. Compared with SHEDs transfected with Si-NC, the mRNA expression of ALP, COL1, and OCN in SHEDs transfected with Si-Axin1 was significantly reduced. In contrast, the expression of these three genes and RUNX2 in SHEDs transduced with LV-Axin1 was significantly higher than that in SHEDs transduced with control lentivirus (LV-NC). Figure 3 Then the present invention verifies the gene expression results by immunoblotting analysis ( Figure 3 As expected, SiRNA-Axin1 and LV-Axin1 led to knockdown and overexpression of Axin1 protein, respectively. In addition, it was found that Si-Axin1 decreased the protein expression of ALP and COL1, while LV-Axin1 increased their expression. The present invention further determined ALP activity by chemical staining of SHED with different levels of Axin1 expression ( Figure 3 C). SHED in which Axin1 expression was reduced by Si-Axin1 transfection had reduced ALP activity. In contrast, LV-Axin1 transduction resulted in increased ALP activity in SHED. In addition, the present invention used Alizarin red staining to characterize the matrix mineralization of SHED after osteogenic induction ( Figure 3 Middle D). After 14 days of osteogenic induction, calcium concentration reflected by Alizarin red staining showed that matrix mineralization was significantly greater in Axin1-overexpressing cells than in control cells. In contrast, Axin1 knockdown resulted in the opposite effect, i.e., matrix mineralization was significantly reduced in Axin1-knockdown cells. Together, these results indicate that Axin1 expression promotes osteogenic differentiation of SHED in vitro.

[0051] Next, the present invention sought to determine whether increased Axin1 expression could promote osteogenic differentiation of SHED in vivo. SHED stably overexpressing Axin1 or control vector via lentiviral transduction were loaded onto Bio-Oss Collagen scaffolds and implanted into the subcutaneous tissue of nude mice (n=3 per group). After 8 weeks, the implants were removed and analyzed by micro-computed tomography (Micro-CT). The ratio of new bone volume to existing tissue volume (BV / TV) was estimated. Figure 4As shown in Figure A, more bone formation was observed in the Axin1 overexpressing SHED implants compared with the control. The BV / TV of the Axin1 overexpressing group was significantly increased compared with the control group. H&E and Masson staining also showed more bone-like structures and collagen deposition in the Axin1 overexpressing implants than in the control implants ( Figure 4 These in vivo results confirmed that Axin1 expression can enhance osteogenic differentiation of SHED.

[0052] Example 3 Exploring the molecular mechanism of Axin1 regulating osteogenesis in SHED 1. Experimental Methods 1. Transcriptome differential analysis A deep sequencing approach was used to identify differentially expressed mRNAs resulting from Axin1 overexpression in cultured SHED. Total RNA samples were prepared from SHED using Trizol reagent (Cat. No. 15596-026; Invitrogen, USA) according to the manufacturer’s instructions. Subsequently, ribosomal RNA (rRNA) was removed from total RNA using Ribo-zero rRNA removal reagent (Epicentre, USA). RNA sequencing libraries were constructed using the NEB Next Ultra II Directional RNA Library Prep Kit (Cat. No. E7760S for Illumina; New England Biolabs) according to the manufacturer’s instructions. The libraries were sequenced on an Illumina HiSeq 4000 system (Illumina, USA). Raw reads during sequencing were filtered by removing low-quality reads and reads containing poly-N or adapters. The reads per kilobase per million mapped (RPKM) values ​​were then calculated for the clean reads in FASTQ format according to the statistical default values ​​provided by Cuffdiff software. A combination of log2 ratio (≥1) and fold change ratio (FDR) value (≤0.001) was used to determine the differential expression of mRNA between groups.

[0053] 2. Bioinformatics analysis Hierarchical clustering analysis of mRNA was performed based on RPKM values ​​using R language software. DAVID (Database for Annotation, Visualization, and Integrated Discovery) was used to analyze the enrichment of differentially expressed mRNAs in the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways.

[0054] 3. FR180204 inhibits ERK FR180204 (Biyuntian, China) is an inhibitor of the ERK1 / 2 pathway. SHED were treated with 3 μM FR180204. The experiment was divided into LV-NC group, LV-Axin1 group, FR180204 group and LV-Axin1+FR180204 group. After 24 hours of treatment, the expression levels of ERK1 / 2 and p-ERK1 / 2 in SHED were detected by Western blotting with reference to step 3 of Example 2, rabbit anti-human ERK1 / 2 (Cell Signaling Technology, USA) and rabbit anti-human p-ERK1 / 2 (Cell Signaling Technology, USA). After 14 days, the mineralization ability of SHED was detected by the above-mentioned Alizarin Red staining and CPC semi-quantitative method.

[0055] 2. Results To explore the molecular mechanism of Axin1 regulating osteogenesis in SHED, we then used RNA sequencing to analyze the differentially expressed genes in Axin1-overexpressing SHED and control cells ( Figure 5 (A). Figure 5 As shown in the volcano plot in B, there were 229 up-regulated genes (78 up-regulated mRNAs) and 217 down-regulated genes (65 down-regulated mRNAs) in SHED with Axin1 overexpression (FDR < 0.001; log2 ratio > 1 or < -1). KEGG pathway analysis revealed that these differentially expressed genes were enriched in multiple osteogenesis-related signaling pathways, such as MAPK, JAK-STAT, and calcium signaling pathways ( Figure 5 Middle C).

[0056] Subsequently, the present invention focused on mRNAs with log2 ratios > 2. On the one hand, the present invention found that the mRNA expression of Axin1 in the LV-Axin1 group was significantly higher than that in the LV-NC group, proving the validity of the SHED of Axin1 overexpression and the sequencing results ( Figure 5 On the other hand, a total of 37 mRNAs were identified under the criterion of log2 ratio > 2, including mRNAs related to calcium signaling pathway (FGFR4, EFCAB9, CASQ1, and TNNC2), MAPK / ERK pathway (ARHGAP15, MAP4K1, ELF3, and MMP10), and VEGF pathway (ANGPTL7) ( Figure 5 In addition, at the protein level, the present invention mined the Peptrack proteomics database (ID: O15169-2) and found that in the MCF10a cell line, src activation led to decreased expression of Axin1 protein and a series of key proteins in the MAPK / ERK pathway ( Figure 5 Middle E).

[0057] KEGG database search showed that the calcium signaling pathway has crosstalk with the MAPK / ERK and VEGF pathways. In the upstream, the activation of the calcium signaling pathway can subsequently activate the MAPK / ERK and VEGF pathways. Figure 5 As shown in DE, the present invention proposes that SHED with overexpression of Axin1 activates the MAPK / ERK pathway and promotes osteogenic differentiation mainly through the calcium signaling pathway. Subsequently, the present invention focused on the MAPK / ERK pathway and verified the results of RNA sequencing analysis by detecting the protein expression of total ERK and p-ERK in SHED with interference or overexpression of AXIN1 by Western blotting. Total protein was extracted from cells transfected or transduced with Si-NC, Si-Axin1, LV-NC or LV-Axin1. The ERK pathway is a type of MAPK pathway, consisting of two subtypes, ERK1 and ERK2, which are highly expressed in osteoblasts and chondrogenic cells. Several studies have shown that the ERK / MAPK pathway promotes osteoblast differentiation and bone formation both in vitro and in vivo. The protein levels of P-ERK1 / 2, ERK1 / 2 and Axin1, as well as the internal reference protein (GAPDH) were detected by Western blot analysis. The results showed that the protein level of phosphorylated ERK1 / 2 (p-ERK1 / 2) in SHED with Axin1 silencing decreased compared with control cells ( Figure 6 In contrast, opposite changes in p-ERK1 / 2 were detected in SHED overexpressing Axin1 ( Figure 6 (B). Notably, the total protein levels of ERK1 / 2 were not affected by Axin1 expression in SHED. These results suggest that Axin1 is involved in the molecular pathways of osteogenesis. In particular, increased Axin1 expression can lead to the activation of the MAPK / ERK pathway.

[0058] To determine whether the ERK1 / 2 pathway is required for Axin1-mediated osteogenic differentiation of SHED, we tested the effect of FR180204, a selective and potent ERK1 / 2 inhibitor. As expected, FR180204 treatment resulted in downregulation of p-ERK in SHED, regardless of Axin1 overexpression ( Figure 6 CD). The p-ERK1 / 2 levels of SHED transduced with LV-NC were reduced after FR180204 treatment. In addition, although the overexpression of Axin1 transduced by lentivirus led to an increase in p-ERK1 / 2 levels, FR180204 treatment completely abolished this effect. After FR180204 treatment, the p-ERK1 / 2 levels in the LV-NC group and the LV-Axin1 group were similar. The present invention obtained consistent results in subsequent functional experiments ( Figure 6Middle D). After two weeks of osteogenic induction, Alizarin red staining showed that FR180204 treatment significantly reduced mineralization in SHED. Axin1 overexpression by LV-Axin1 transduction increased mineralization, while FR180204 treatment significantly reduced this increase ( Figure 6 These results suggest that Axin1 expression promotes osteogenic differentiation of SHED at least in part through ERK1 / 2 activation.

[0059] In summary, the present invention explored the effect of Axin1 expression on the cell characteristics of MSCs (SHED), especially its osteogenic differentiation potential. The present invention isolated SHED and used lentivirus containing the Axin1 gene or small interfering RNA targeting Axin1 to regulate the expression of Axin1. The present invention first studied the effect of Axin1 expression on the proliferation, migration and mesenchymal characteristics of SHED by CCK8 method, Transwell experiment and flow cytometry (FCM), respectively. In addition, the present invention also performed alkaline phosphatase (ALP) activity detection, Alizarin red staining, Western blotting (WB), real-time quantitative polymerase chain reaction (qRT-PCR) and in vivo osteogenesis experiments to determine the effect of Axin1 expression on SHE osteogenesis. The present invention found that overexpression and knockdown of Axin1 did not affect the proliferation and MSC characteristics of SHED. However, overexpression of Axin1 promoted the migration of SHED, and this effect could be weakened by Axin1 silencing. Overexpression of Axin1 promoted the osteogenic differentiation of SHED, while its knockdown showed the opposite effect. Mechanistically, overexpression of Axin1 significantly increased the expression level of phosphorylated ERK1 / 2, thereby promoting the osteogenic differentiation of SHED. Treatment with chemical inhibitors of ERK1 / 2 eliminated the promoting effect of Axin1 overexpression on the osteogenic differentiation of SHED. This indicates that Axin1 plays a role as a key regulator of osteogenic differentiation in SHED through the ERK1 / 2 pathway.

Claims

1. Application of Axin1 in positively regulating the osteogenic differentiation of dental mesenchymal stem cells.

2. Application of Axin1 in the preparation of products that promote the osteogenic differentiation of dental mesenchymal stem cells.

3. Use of preparations that promote Axin1 overexpression in the preparation of products that promote osteogenic differentiation of dental mesenchymal stem cells.

4. The use according to claim 2 or 3, characterized in that: The product overexpresses the Axin1 gene to increase the expression level of osteogenesis-related genes, thereby promoting the osteogenic differentiation of dental mesenchymal stem cells.

5. The application according to claim 4, characterized in that: The osteogenesis-related genes include ALP, RUNX2, COL1 and OCN.

6. The use according to claim 2 or 3, characterized in that: The product promotes the osteogenic differentiation of dental mesenchymal stem cells by overexpressing the Axin1 gene and increasing the expression level of phosphorylated ERK1 / 2.

7. The use according to any one of claims 1 to 3, characterized in that: The dental mesenchymal stem cells are deciduous tooth pulp stem cells separated from deciduous teeth.

8. A method for promoting osteogenic differentiation of dental mesenchymal stem cells, characterized in that: Dental mesenchymal stem cells were treated with agents that promoted Axin1 overexpression and then cultured for osteogenic differentiation.

9. The method according to claim 8, characterized in that: The preparation for promoting overexpression of Axin1 is an expression vector containing Axin1 gene.

10. The method according to claim 8, characterized in that: The vector is a lentiviral vector.

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