Application of Axin1 in regulating osteogenic differentiation of primary dental pulp stem cells

Abstract

The present invention discloses the application of Axin1 in regulating the osteogenic differentiation of deciduous dental pulp stem cells. The research of 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 attenuated 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 a chemical inhibitor of ERK1 / 2 can eliminate the promoting effect of Axin1 overexpression on the osteogenic differentiation of SHED, indicating that Axin1 acts as a key regulator of osteogenic differentiation in SHED through the ERK1 / 2 pathway. The research results of the present invention show that Axin1 can be used as a potential new target for applying SHED to treat diseases involving bone regeneration.

Description

Technical Field

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

[0002] Regenerative medicine has great potential for patients with bone problems, which have a significant negative impact on the quality of life of patients 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 grafts have many limitations, such as limited bone resources, low osteoinductive and bone integration capabilities, and immune rejection of allogeneic grafts. In recent years, a method of regenerative treatment using tissue-engineered bone (TEB) composed of mesenchymal stem cells (MSCs) + scaffold material + cytokines has been widely studied. This method can greatly improve the safety and effectiveness of bone regeneration treatment and avoid processes such as invasive surgery. Currently, it is considered 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), which are derived from the dental pulp tissue of human exfoliated deciduous teeth, are an ideal source for cell therapy due to their rich tissue source and easy accessibility. 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 have 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 reported previously that the cytokine secretion ability affects the osteogenic differentiation of MSCs. In addition, compared with dental pulp stem cells (DPSCs) and bone marrow mesenchymal stem cells (BMSCs), SHED have higher proliferation and osteogenic activity because they can produce higher levels of osteocalcin (OCN) and alkaline phosphatase (ALP), and express more basic fibroblast growth factor (bFGF) and bone morphogenetic protein 2 (BMP2) after differentiation. Many preclinical studies have confirmed that SHED are particularly suitable for bone regeneration.

[0004] Axin is a cytoskeletal protein that regulates the formation of body axes during embryonic development of organisms. By associating with multiple signaling pathways such as Wnt, JNK, TGF-β, and AMP-activated protein kinase (AMPK), it affects biological processes such as cell proliferation, differentiation, apoptosis, and carcinogenesis, and is an important gene that controls processes such as cell proliferation and differentiation and the cell cycle. It has been confirmed that there are 2 members in the Axin family: Axin1 and its homolog Axin2 (Axil). They have the same domain and functional consistency. Among them, Axin1 is ubiquitously expressed in tissues, while Axin2 is only expressed in specific tissues and developmental stages.

[0005] Recent studies on Axin in bone tissue have gradually attracted the attention of scholars. It has been reported that knockdown of Axin1 can inhibit the apoptosis of osteoblasts induced by Porphyromonas gingivalis lipopolysaccharide. In addition, inhibition of Axin1 in osteoprogenitor cells has been found to lead to postnatal bone growth defects due to the inhibition of osteoclast formation. Moreover, it has been demonstrated that deletion of Axin1 in condylar chondrocytes causes degeneration of mouse condylar cartilage by activating the β-catenin and ERK1 / 2 signaling pathways. However, there is a lack of relevant research on the effect of Axin1 on the function of MSCs, especially its functional role, expression level, and molecular regulatory mechanism during the osteogenic differentiation of MSCs. In particular, the effect of Axin1 on the biological characteristics and osteogenic differentiation potential of SHED has not been reported yet. 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 research of 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 attenuated by Axin1 silencing. Overexpression of Axin1 promotes the osteogenic differentiation of SHED, while its knockdown shows the opposite effect. It is indicated that Axin1 can be used as a potential new target for applying SHED to treat diseases involving bone regeneration.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] The present invention aims to explore the effect of Axin1 expression on the cell characteristics of MSCs (SHED), especially its osteogenic differentiation potential. By studying SHED, an attempt is made 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 applying SHED to treat diseases involving bone regeneration.

[0009] Specifically, the present invention isolated SHED and used lentivirus containing the Axin1 gene or small interfering RNA against Axin1 to regulate the expression of Axin1. The present invention first studied the effects of Axin1 expression on the proliferation, migration and mesenchymal characteristics of SHED by CCK8 method, Transwell assay and flow cytometry (FCM) respectively. In addition, the present invention also performed alkaline phosphatase (ALP) activity assay, alizarin red staining, Western blotting (WB), real-time quantitative polymerase chain reaction (qRT-PCR) and in vivo osteogenesis assay to determine the effects of Axin1 expression on the osteogenesis of SHE. 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 attenuated 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 a chemical inhibitor of ERK1 / 2 could eliminate the promoting effect of Axin1 overexpression on the osteogenic differentiation of SHED. It is indicated that Axin1 plays a role as a key regulator of osteogenic differentiation in SHED through the ERK1 / 2 pathway.

[0010] Therefore, the present invention provides the application of Axin1 in positively regulating the osteogenic differentiation of dental mesenchymal stem cells.

[0011] The present invention also provides the application of Axin1 as a target in the preparation of products for promoting the osteogenic differentiation of dental mesenchymal stem cells.

[0012] The present invention also provides the application of a preparation for promoting Axin1 overexpression in the preparation of products for promoting the osteogenic differentiation of dental mesenchymal stem cells.

[0013] Furthermore, the product promotes the osteogenic differentiation of dental mesenchymal stem cells by overexpressing the Axin1 gene and increasing the expression levels of osteogenesis-related genes.

[0014] Furthermore, the osteogenesis-related genes include ALP, RUNX2, COL1 and OCN.

[0015] 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.

[0016] Furthermore, the product is a drug for treating bone defects.

[0017] The present invention also provides a method for promoting the osteogenic differentiation of dental mesenchymal stem cells. The method is to first treat the dental mesenchymal stem cells with a preparation that promotes the overexpression of Axin1, and then perform osteogenic induction differentiation culture on the treated dental mesenchymal stem cells.

[0018] Furthermore, the preparation that promotes the overexpression of Axin1 is an expression vector containing the Axin1 gene.

[0019] Preferably, the vector is a lentiviral vector.

[0020] Furthermore, the osteogenic induction differentiation culture is to perform induction culture on the treated dental mesenchymal stem cells in an osteogenic differentiation induction medium.

[0021] As a preferably feasible implementation manner, the method for promoting the osteogenic differentiation ability of dental mesenchymal stem cells includes the following steps:

[0022] S1. Obtain dental mesenchymal stem cells and perform passage culture;

[0023] S2. Construct an expression vector for overexpressing the Axin1 gene;

[0024] S3. Transfect the constructed expression vector for overexpressing the Axin1 gene into dental mesenchymal stem cells, and then perform induction culture on the transfected dental mesenchymal stem cells in an osteogenic differentiation induction medium.

[0025] 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 glycerophosphate to DMEM.

[0026] Furthermore, the above-mentioned dental mesenchymal stem cells include, but are not limited to, dental pulp stem cells isolated from deciduous teeth.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention explores the effect of Axin1 expression on the cell characteristics of MSCs (SHED), especially its osteogenic differentiation potential. The research of this 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 attenuated 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 a chemical inhibitor of ERK1 / 2 can eliminate 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 research results of this invention show that Axin1 can be used as a potential new target for treating diseases involving bone regeneration by applying SHED. Description of the Drawings

[0029] Figure 1 Results of the effect of Axin1 expression on the proliferation and migration of SHED. Among them, Figure 1 A shows the results of the expression level of Axin1 mRNA after transfection or transduction of SHED with siRNA targeting Axin1 or lentiviral particles overexpressing Axin1; B shows the expression of Axin1 protein in SHED after transfection of SHED with siRNA targeting Axin1 or transduction of SHED with lentiviral particles overexpressing Axin1. The histogram is the quantitative result of the band intensity; C shows the results of inverted fluorescence microscopy of SHED two days after transfection with lentivirus overexpressing Axin1; D shows the results of the effect of Axin1 silencing or overexpression on the migration of SHED, and E shows the effect of Axin1 silencing (left) or overexpression (right) on the proliferation of SHED; 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.

[0030] Figure 2 Results of the effect of Axin1 overexpression on the expression of cell surface marker antigens of SHED. Among them Figure 2 A shows the flow cytometry results of cell surface marker antigens of SHED after transduction of SHED with negative control lentiviral particles (LV-NC); B shows the flow cytometry results of cell surface marker antigens of SHED after transduction of SHED with lentiviral particles containing Axin1 transgene (LV-Axin1) to overexpress Axin1.

[0031] Figure 3 Results of the effect of Axin1 expression on the osteogenic effect of SHED in vitro. Among them, Figure 3 A shows the RT-PCR detection results of osteogenic genes after osteogenic induction of SHED cultured for 7 days; B shows the Western blot results of osteogenic genes after osteogenic induction of SHED cultured for 7 days, and the histograms (middle and right) show the quantitative results of band intensity; C shows the results of the effect of Axin1 expression on ALP activity in SHED, and the histogram is the quantitative result of ALP; D shows the results 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.

[0032] Figure 4 Results of the effect of Axin1 expression on the osteogenic effect of SHED in vivo. Among them, Figure 4 A shows the reconstructed three-dimensional micro-computed tomography (micro-CT) images (left figure) of representative tissue-engineered bone constructs of implants loaded with SHED transduced with LV-Axin1 or LV-NC, and the ratio of new bone volume (BV) to original existing tissue volume (TV) (BV / TV) of bone constructs of implants loaded with SHED transduced with LV-Axin1 and LV-NC (right figure, n = 3 per group), data are expressed as mean ± standard deviation, *P<0.05; B shows the results of hematoxylin-eosin (H&E) staining and Masson staining of osteocalcin of implants with different Axin1 expressions.

[0033] Figure 5 Results of RNA sequencing analysis of SHED with overexpressed Axin1 and control cells. Among them, Figure 5 A shows the hierarchical clustering results of differentially expressed mRNAs in SHED with overexpressed Axin1 and control cells; B shows the volcano plot of differentially expressed mRNAs between SHED with overexpressed Axin1 and control SHED; C shows the functional classification results of differentially expressed mRNAs induced by overexpressed Axin1 in Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis; D shows the sequencing results; E shows the results of Peptrack proteomics database.

[0034] Figure 6 Results of the functional verification that Axin1 activates the ERK1 / 2 pathway to promote the osteogenic differentiation of SHED. Among them, Figure 6In A, the effect of Axin1 knockdown on ERK phosphorylation is shown; in B, the effect of Axin1 overexpression on ERK phosphorylation is shown. Left: protein immunoblotting images; right: semi-quantitative results of the p-ERK / ERK ratio in SHED with Axin1 silencing or overexpression and control cells. In C, the effect of the ERK inhibitor FR180204 on ERK phosphorylation in SHED with and without Axin1 overexpression is shown. Left: protein immunoblotting images; right: quantitative analysis results of the p-ERK / ERK ratio. In D, the effect of ERK inhibition on matrix mineralization in SHED with and without Axin1 overexpression is shown. Left: alizarin red staining images of calcified nodules; right: histograms showing the quantitative results of alizarin red staining by spectrophotometry. The 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 the Axin1 transgene. ERK: extracellular signal-regulated kinase. *P<0.05; **P<0.01; ***P<0.001. Detailed implementation manners

[0035] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments 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 technical field.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0037] Example 1 Effect of Axin1 on the biological properties of stem cells from human exfoliated deciduous teeth

[0038] I. Experimental methods

[0039] 1. Isolation and culture of SHED

[0040] SHED was isolated from the retained deciduous teeth of healthy children aged 6 - 12 years. This study was approved by the Medical Ethics Committee of the Stomatological Hospital Affiliated to Guangzhou Medical University (approval number: KY2019008), and written informed consent was obtained from the parents of the patients. Under sterile conditions, dental pulp tissues were isolated and minced, digested with 3 g / L collagenase I (Gibco, USA) and 4 g / L dispase for 30 minutes, centrifuged for 5 minutes, and the collected cells were inoculated into a T25 culture flask containing DMEM medium (Gibco, USA) with 0% fetal bovine serum (Gibco, USA) and 1% penicillin / streptomycin (Gibco, USA) for culture. When the cells reached 80% confluence, they were digested with trypsin and continuously passaged. As described below, the positive and negative surface markers of SHED as MSCs (CD73+ / CD90 + / CD105 + / CD34 − / CD45 − ), and its osteogenic differentiation ability (osteogenic induction). SHED of passages 3-6 were collected for subsequent experiments.

[0041] 2. Overexpression and knockdown of Axin1 in SHED

[0042] SHED were transduced with recombinant lentivirus containing the human Axin1 gene to overexpress Axin1, or transfected with Axin1 siRNA to knockdown the expression of Axin1. For overexpression, Axin1-GFP lentiviral particles (LV-Axin1) and negative control empty vector-GFP control particles (LV-NC) (Shanghai Genechem Co., Ltd., China) were used and treated at a specified multiplicity of infection in serum-free medium. After incubation with the virus for 6 hours, the medium was replaced with fresh complete medium. The transduction efficiency was examined under a fluorescence microscope 48 hours later, and the expression of Axin1 was determined by real-time polymerase chain reaction (RT-PCR). For knockdown of Axin1, SHED were seeded in 6-well plates, and when the cells reached 75%-90% confluence, siRNA was transfected using Lipofectamine 3000 (Invitrogen, USA). Predesigned and validated siRNA targeting Axin1 (Si-Axin1, target sequence: GCATCGTTGTGGCGTACTA) and control nontargeting siRNA (Si-NC, F: 5'-UUCUCCGAACGUGUCACGUTT-3', R: 5'-ACGUGACACGUUCGGAGAATT-3') were purchased from Ribobio (Guangzhou, China). After incubation for 72 hours, RT-PCR was performed to detect the expression of Axin1.

[0043] 3. Flow cytometry analysis of cell surface markers

[0044] The 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). For cells treated with Axin1 siRNA or Si-control, CD34 was labeled with fluorescein isothiocyanate (FITC), and for cells treated with Axin1-GFP lentiviral particles or LV-NC, CD34 was labeled with PE.

[0045] 4. CCK8 assay for cell proliferation

[0046] Cell proliferation was evaluated using a CCK8 kit (Dojindo, Japan) according to the manufacturer's instructions. SHED were seeded into 96-well plates at a density of 1×10 3 cells per well. Cell proliferation was detected at 1, 3, 5, and 7 days after seeding. After incubation with the 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.

[0047] 5. Transwell assay for cell migration

[0048] The transfected SHED were seeded into the upper chamber of a Transwell at a density of 5×10 4 cells per well. Cells growing in the upper chamber were cultured in serum-free medium, while complete medium was added to the lower chamber of the Transwell. After incubation for 48 hours, 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 minutes and stained with 0.1% crystal violet (Shanghai Bogu, China). Crystal violet-positive cells in five random fields were photographed under an inverted microscope (Leica Instruments, USA). The cell density was analyzed using ImageJ software.

[0049] 6. RT-PCR assay for the expression level of Axin1 gene

[0050] SHED were seeded into each well at a density of 1×10 5Cells were seeded in 6-well plates at a density of. Total RNA was extracted from the cultured cells using TRIzol reagent (Invitrogen, USA). mRNA (500 ng) was reverse-transcribed into cDNA using the PrimeScript RT Master Mix kit (Takara, Japan). cDNA was subjected to RT-PCR using SYBR® Premix Ex Taq TM (Takara, Japan). The primer sequences for RT-PCR are shown in Table 1. RT-PCR was performed using an ABI Step-OnePlus real-time PCR system for 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. Reaction system: SYBR® Premix Ex Taq TM 10 μL, PCR Forward Primer (10 μM) 0.4 μL, PCR Reverse Primer (10 μM) 0.4 μL, ROX Reference Dye (50×) 0.4 μL, cDNA 2.0 μL, RNase Free Water 6.8 μL, with a total volume of 20 μL. GAPDH was used as an internal control. The relative expression levels of target genes were calculated using the 2-ΔΔCt method.

[0051] Table 1 Sense and antisense primers for real-time reverse transcription polymerase chain reaction (RT-PCR)

[0052]

[0053] 7. Statistical analysis

[0054] All experiments were performed in triplicate. Experimental data are expressed as the mean ± standard deviation. T-test analysis was performed using SPSS 20.0 statistical software, and graphs were plotted using GraphPad Prism. A P < 0.05 was considered statistically significant.

[0055] II. Results

[0056] To investigate the function of Axin1 in SHED, the present invention used siRNA targeting Axin1 and lentiviral particles (LV-Axin1) containing the Axin1 cDNA sequence to regulate the expression of Axin1. To silence or overexpress Axin1, small interfering RNA (siRNA) targeting Axin1 was transfected into SHED or SHED was transduced with lentiviral particles (LV), respectively. 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, 3) were tested, and Si-Axin1-1 was selected for subsequent experiments, as Figure 1 shown in Figure 1 Figure A and Figure 1 Figure B. After 3 days of treatment, the expression of Axin1 was successfully silenced by siRNA transfection and enhanced by lentiviral transduction. Using the marker GFP transgenic in the lentiviral construct, the present invention traced the lentivirus-mediated gene overexpression. As early as 2 days after lentiviral transduction, the fluorescent protein was highly expressed in SHED ( Figure 3 Figure C). Knockdown and overexpression of Axin1 were continuously achieved during the experiment ( Figure 1 Figure B and data not shown). Axin1 is a scaffold protein. The present invention further analyzed the effect of its expression on the migration of SHED using Transwell assays. Interestingly, compared with cells treated with non-targeting control siRNA (Si-NC), Axin1 silencing significantly reduced the migration of SHED ( Figure 1 Figure D). In contrast, Axin1 overexpression by lentiviral transduction led to a significant increase in SHED migration ( Figure 1 Figure D). Through cell viability assays, it was found that Axin1 silencing or overexpression had no effect on the proliferation of SHED ( Figure 2 Figure A and Figure 2 Figure B). SHED belongs to MSCs. To characterize SHED 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 SHED expressed CD73, CD90, and CD105, while CD19, CD34, and CD45 were hardly detectable (less than 1% of SHED). Notably, Axin1 overexpression by lentiviral transduction had no effect on the expression of these MSC surface biomarkers, indicating that the increase in Axin1 expression does not affect the stemness of SHED (

[0057] In summary, interfering with and overexpressing Axin1 do not affect the stemness and proliferation ability of SHED, and SHED still possesses the characteristics of mesenchymal stem cells. After interfering with Axin1, the migration ability of SHED is significantly reduced, while after overexpressing Axin1, the migration ability of SHED is significantly enhanced, indicating that positive regulation of Axin1 can promote the migration of SHED.

[0058] Example 2 Effect of Axin1 Expression on Osteogenic Differentiation of SHED

[0059] I. Experimental Methods

[0060] 1. Osteogenic Differentiation

[0061] The 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 glycerophosphate (all purchased from Shanghai Sangon Biotech Co., Ltd.) to 90 mL of DMEM and stored at 4°C. During the in vitro osteogenic differentiation process, SHED cells after interfering with or overexpressing Axin1 were seeded in 6-well plates and induced with the osteogenic induction medium, and the medium was changed every 2 - 3 days.

[0062] 2. Detection of mRNA Expression Levels of Osteogenic Genes in SHED by RT-PCR

[0063] Performed according to the experimental method step 6 of Example 1 to detect the gene expressions of runt-related transcription factor 2 (Runx2), type I collagen (COL1), alkaline phosphatase (ALP), and osteocalcin (OCN).

[0064] 3. Western Blot Analysis

[0065] The transfected SHED were treated with RIPA buffer (Thermo Fisher, USA) and centrifuged at 12,000 rpm for 15 minutes at 4 °C to collect the protein supernatant. The total protein concentration of each sample was measured using a BCA kit (Thermo Fisher, USA). Protein samples (20 μg) were separated by 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) at 37 °C for 2 hours and then incubated overnight at 4 °C with different primary antibodies: rabbit anti-human GAPDH (Cell Signaling 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 minutes, the membrane was incubated with HRP-conjugated affinity-purified goat anti-rabbit IgG for 2 hours at room temperature. Protein bands were visualized using a Western blot imaging system (Bio-Rad, USA).

[0066] 4. Alkaline phosphatase activity detection

[0067] The transfected SHED were seeded in 48-well plates at a density of 1×10 4 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 ALP activity was normalized to the total protein content relative to the control group. For ALP staining, the transfected SHED were washed with PBS and fixed with 4% paraformaldehyde for 30 minutes. After washing three times with PBS, the cells were stained using an NBT / BCIP staining kit (Beyotime, China) according to the manufacturer's instructions. Images were taken under a microscope (Leica Instruments, USA).

[0068] 5. Alizarin red staining and quantification

[0069] The transfected SHED were seeded in each well at a density of 2×10 4Cells were seeded at a density of [number] cells per well in a 48-well culture plate. After 14 days of culture, the cells were fixed with 95% ethanol at room temperature (RT) for 30 minutes, rinsed three times with double-distilled water, stained with 5% alizarin red (pH = 4.2, Sigma Aldrich, USA) for 5 - 10 minutes, washed repeatedly with double-distilled water, and then dried at room temperature. The dried culture plates were photographed under a stereomicroscope (Leica Instruments, USA). For quantification of calcified nodules, alizarin red dissolved in 10% cetylpyridinium chloride (CPC, Beyotime, China) was added to the culture plates. After incubation at room temperature for 1 hour, 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.

[0070] 6. Osteogenesis assay and micro-computed tomography (Micro-CT) analysis

[0071] SHED with overexpressed Axin1 and the control group 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 hours were implanted subcutaneously into the back 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 Experimental Ethics Committee of the Guangdong Provincial Quality Supervision and Inspection Station for Medical Devices (Approval No.: 2021012001). Micro-CT analysis was performed using a high-resolution Inveon Micro-CT (Bruker, Germany) with the X-ray source set at 71 kV, the tube current at 100 μA, and the scanning resolution at 10 μm. Image slices were reconstructed using Micro-CT image analysis software (Inveon Research Workplace). The ratio of new bone volume to the volume of existing tissue (BV / TV) was calculated.

[0072] 7. Histological and histomorphometric analysis

[0073] Tissues were decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 4 weeks, dehydrated, and paraffin-embedded. Tissue sections were prepared and stained with hematoxylin and eosin (H&E) or Masson's trichrome staining. Ten random fields of each section were photographed under a microscope.

[0074] 8. Statistical analysis

[0075] All experiments were performed in triplicate, and all data are expressed as mean ± standard deviation. T-test analysis and One-Way ANOVA analysis were performed using SPSS 20.0 statistical software, and graphs were plotted using GraphPad Prism.P < 0.05 The difference was statistically significant.

[0076] II. Results

[0077] SHED 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 after 7 days of osteogenic induction, including ALP, RUNX2, COL1, and OCN. Compared with SHED transfected with Si-NC, the mRNA expression of ALP, COL1, and OCN in SHED transfected with Si-Axin1 was significantly decreased. In contrast, the expression of these three genes and RUNX2 in SHED transduced with LV-Axin1 was significantly higher than that in SHED transduced with the control lentivirus (LV-NC) ( Figure 3 in A). Then the present invention verified the gene expression results by immunoblot analysis ( Figure 3 in B). 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 the ALP activity by chemical staining of SHED with different levels of Axin1 expression ( Figure 3 in C). SHED with reduced Axin1 expression by Si-Axin1 transfection had reduced ALP activity. In contrast, LV-Axin1 transduction led to an increase in 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 in D). After 14 days of osteogenic induction, the calcium concentration reflected by alizarin red staining showed that the matrix mineralization in Axin1 overexpressing cells was significantly more than that in control cells. In contrast, Axin1 knockdown led to the opposite effect, that is, significantly reduced matrix mineralization in Axin1 knockdown cells. In summary, these results indicate that Axin1 expression promoted the osteogenic differentiation of SHED in vitro.

[0078] Next, the present invention attempted to determine whether an increase in Axin1 expression could promote the osteogenic differentiation of SHED in vivo. SHED stably overexpressing Axin1 or the control vector by lentiviral transduction was 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 the volume of new bone to the volume of existing tissue (BV / TV) was estimated. As Figure 4As shown in A, compared with the control, there was more bone formation in the SHED implants with Axin1 overexpression. The BV / TV in the Axin1 overexpression group was significantly increased compared with the control group. H&E and Masson staining also showed more osteoid structures and collagen deposition in the Axin1 overexpression implants than in the control implants ( Figure 4 in B). These in vivo results confirmed that Axin1 expression could enhance the osteogenic differentiation of SHED.

[0079] Example 3 Exploring the molecular mechanism of Axin1 regulating osteogenesis in SHED

[0080] I. Experimental methods

[0081] 1. Transcriptome differential analysis

[0082] Deep sequencing was used to identify differentially expressed mRNAs caused by 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 the total RNA using Ribo-zero rRNA Removal Reagent (Epicentre, USA). An RNA sequencing library was 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 library was sequenced on an Illumina Hiseq 4000 system (Illumina, USA). The raw reads during the sequencing process were filtered by removing low-quality reads and reads containing poly-N or adapters. Subsequently, the reads per kilobase per million mapped reads (RPKM) values of the clean reads in FASTQ format were calculated according to the statistical defaults provided by the Cuffdiff software. The combined use of log2 ratio (≥1) and fold change ratio (FDR) value (≤0.001) was used to determine the differential expression of mRNAs between groups.

[0083] 2. Bioinformatics analysis

[0084] Hierarchical clustering analysis of mRNAs was performed using R language software based on the RPKM values. 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.

[0085] 3. FR180204 inhibits ERK

[0086] FR180204 (Beyotime, China) is an inhibitor of the ERK1 / 2 pathway. SHED was treated with 3 μM FR180204. The experiments were 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 according to the steps of Example 2, step 3, with 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 alizarin red staining and CPC semi-quantitative method.

[0087] II. Results

[0088] To explore the molecular mechanism of Axin1 regulating osteogenesis in SHED, the present invention then used RNA sequencing to analyze the differentially expressed genes in Axin1-overexpressing SHED and control cells ( Figure 5 in A). As shown in the volcano plot of Figure 5 in B, there were 229 upregulated genes (78 upregulated mRNAs) and 217 downregulated genes (65 downregulated mRNAs) in Axin1-overexpressing SHED (FDR < 0.001; log2 ratio > 1 or < -1). Through KEGG pathway analysis, it was found that these differentially expressed genes were enriched in multiple osteogenesis-related signaling pathways, such as MAPK, JAK-STAT and calcium signaling pathways ( Figure 5 in C).

[0089] Subsequently, the present invention focused on mRNAs with a log2 ratio > 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, demonstrating the effectiveness of Axin1-overexpressing SHED and the sequencing results ( Figure 5 in D left). On the other hand, a total of 37 mRNAs were identified under the criterion of a log2 ratio > 2, including mRNAs related to the calcium signaling pathway (FGFR4, EFCAB9, CASQ1 and TNNC2), MAPK / ERK pathway (ARHGAP15, MAP4K1, ELF3 and MMP10) and VEGF pathway (ANGPTL7) ( Figure 5In the right panel of Figure D). 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 a decrease in the expression of Axin1 protein and a series of key proteins in the MAPK / ERK pathway ( Figure 5 In the right panel of Figure E).

[0090] KEGG database search showed that there was crosstalk between the calcium signaling pathway and the MAPK / ERK and VEGF pathways. Upstream, activation of the calcium signaling pathway could subsequently activate the MAPK / ERK and VEGF pathways. As shown in Figures D-E of the present invention, SHED with overexpressed Axin1 mainly activated the MAPK / ERK pathway and promoted osteogenic differentiation 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 by Western blotting after interfering with or overexpressing AXIN1. Total proteins were 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 and consists of two subtypes, ERK1 and ERK2, which are highly expressed in osteoblasts and chondrocyte progenitors. Multiple studies have shown that the ERK / MAPK pathway promotes osteoblast differentiation and bone formation both in vivo and in vitro. The protein levels of P-ERK1 / 2, ERK1 / 2, Axin1 and the internal reference protein (GAPDH) were detected by Western blotting analysis. The results showed that the protein level of phosphorylated ERK1 / 2 (p-ERK1 / 2) decreased in SHED with silenced Axin1 compared with control cells ( Figure 5 In the left panel of Figure A). Conversely, the opposite change in p-ERK1 / 2 was detected in SHED with overexpressed Axin1 ( Figure 6 In the right panel of Figure B). Notably, the total protein level of ERK1 / 2 was not affected by the expression of Axin1 in SHED. These results indicate that Axin1 is involved in the molecular pathway of osteogenesis. In particular, an increase in the expression of Axin1 can lead to the activation of the MAPK / ERK pathway. Figure 6 To determine whether the ERK1 / 2 pathway is essential for Axin1-mediated osteogenic differentiation of SHED, the present invention tested the effect of FR180204 (a selective and effective ERK1 / 2 inhibitor). As expected, treatment with FR180204 led to a downregulation of p-ERK in SHED regardless of whether Axin1 was overexpressed (

[0091] Figure 6 Figure 6In (C-D). After treatment with FR180204, the level of p-ERK1 / 2 in SHED transduced with LV-NC decreased. In addition, although overexpression of Axin1 by lentiviral transduction led to an increase in the level of p-ERK1 / 2, treatment with FR180204 completely abolished this effect. After treatment with FR180204, the levels of p-ERK1 / 2 in cells of the LV-NC group and the LV-Axin1 group were similar. The present invention obtained consistent results in subsequent functional experiments ( Figure 6 In (D). After two weeks of osteogenic induction, alizarin red staining showed that treatment with FR180204 significantly reduced mineralization in SHED. Overexpression of Axin1 by LV-Axin1 transduction increased mineralization, while treatment with FR180204 significantly reduced this increase ( Figure 6 In (D). These results indicate that Axin1 expression promoted osteogenic differentiation of SHED at least partially through ERK1 / 2 activation.

[0092] 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 against Axin1 to regulate the expression of Axin1. The present invention first studied the effects of Axin1 expression on the proliferation, migration and mesenchymal characteristics of SHED by CCK8 assay, Transwell assay and flow cytometry (FCM), respectively. In addition, the present invention also performed alkaline phosphatase (ALP) activity assay, alizarin red staining, Western blotting (WB), real-time quantitative polymerase chain reaction (qRT-PCR) and in vivo osteogenesis experiment to determine the effect of Axin1 expression on osteogenesis of SHE. 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 attenuated 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, thus promoting the osteogenic differentiation of SHED. Treatment with a chemical inhibitor of ERK1 / 2 abolished the promoting effect of Axin1 overexpression on the osteogenic differentiation of SHED. It is indicated that Axin1 plays a role as a key regulator of osteogenic differentiation in SHED through the ERK1 / 2 pathway.

Claims

1. Application of overexpression of human Axin1 gene in positively regulating osteogenic differentiation of dental mesenchymal stem cells, wherein the dental mesenchymal stem cells are deciduous tooth pulp stem cells isolated from deciduous teeth.

2. Use of human Axin1 gene in the preparation of a product for promoting osteogenic differentiation of dental mesenchymal stem cells, wherein the dental mesenchymal stem cells are deciduous tooth pulp stem cells isolated from deciduous teeth.

3. Use of a preparation that promotes overexpression of the human Axin1 gene in the preparation of a product that promotes osteogenic differentiation of dental mesenchymal stem cells; the preparation that promotes overexpression of the human Axin1 gene is an overexpression vector containing the human Axin1 gene; the dental mesenchymal stem cells are deciduous tooth pulp stem cells isolated from deciduous teeth.

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

5. The use 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 human Axin1 gene and increasing the expression level of phosphorylated ERK1 / 2.

7. A method for promoting osteogenic differentiation of dental mesenchymal stem cells, characterized in that: Dental mesenchymal stem cells are treated with a preparation that promotes overexpression of the human Axin1 gene, and then cultured for osteogenic differentiation induction; the preparation that promotes overexpression of the human Axin1 gene is an overexpression vector containing the human Axin1 gene; and the dental mesenchymal stem cells are deciduous tooth pulp stem cells separated from deciduous teeth.

8. The method according to claim 7, characterized in that: The vector is a lentiviral vector.

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