| Background and objectiveBone marrow mesenchymal stem cells(MSCs)are potential pluripotent cells,which are capable of self renewal and multipotential differentiation,and can differentiate bone,fat,cartilage and muscle.In recent years,more and more data have shown that Wnt signaling pathway can regulate osteoblast differentiation in bone marrow mesenchymal stem cells.The effect of Wnt gene on the differentiation of osteogenic differentiation suggests that different Wnt genes and their transcription factors may have different effects on the differentiation of stem cells,while different Wnt genes and their transcription factors show different patterns of expression in bone differentiation.In order to further explore the role of Wnt7a gene in osteoblast differentiation of bone marrow mesenchymal stem cells,we overexpressed and silenced Wnt7a gene in bone marrow mesenchymal stem cells,and explored the effect of Wnt7a gene on osteoblast differentiation in bone marrow mesenchymal stem cells.Methods1.Isolation and identification of bone marrow mesenchymal stem cells from people.The human bone marrow tissue was taken for primary culture.Epics XL flow cytometry was used to detect the expression of the marker antigen of human bone marrow mesenchymal stem cells.The main antigens used included 5 kinds:CD44,CD90,CD105,CD34 and CD45.The immunofluorescence staining method was used to detect the positive expression of Ki67 cell nuclear antibody in the cells in order to evaluate the proliferation ability of the isolated cells.In the experiment,we used mouse IgG2a(Santa Cruz)as a parallel control against Ki-67 mouse McAb,that is,positive control,and FITC-conjugated goat anti-mouse antibody as negative control.2.In vitro induced differentiation and expression of Wnt7aAfter osteogenesis,alizarin red was used to stain the cells,and the markers of osteogenic differentiation(OCN,OPN)mRNA were detected by fluorescence quantitative RT-PCR.After induction of differentiation with fat induced medium,the cells were stained with oil red staining.The markers of adipose differentiation(PPAR gamma,LPL)mRNA were detected.The Wnt7a mRNA transcriptional and protein expression of the induced cells were detected respectively.3.Lentivirus system silencing and overexpressing Wnt7a geneThe gene transfection system of lentivirus silencing and overexpressing Wnt7a gene was constructed respectively.The induction of osteogenic differentiation was carried out respectively.Detection of the above markers of bone differentiation and the transcription and expression of RUNX2 and OSX.4.Wnt7a regulates bone differentiation in bone marrow mesenchymal stem cells through expression of RUNX2.Amplify and purify TCF1 binding site fragment(located at the upstream of RUNX2 gene exon-1056 to-1062 BP),and insert the purified DNA fragment into SacI/XhoI location of pGL3-control plasmid.The activity of the region was measured by luciferase activity.CHIP assay was used to detect the combination of RUNX2,TCF1 and Promoter after the Wnt7a gene was silenced and the overexpressed Wnt7a gene was differentiated into bone.ChIP assay kit is used to carry out the related experiments,and the specific steps are operated according to the experimental scheme.Results1.Isolation and identification of bone marrow mesenchymal stem cells from people.The morphology of the cultured cells at different stages was observed under the microscope.The primary cells were isolated and cultured for 48h,which showed cell wall growth and cell separation and increment.After 6 passages,at the 7d of culture,the cells adhered to the wall grew evenly and well.The single cells were spindle/fibroblast,and no obvious morphological variation was observed.The morphology of the bone marrow mesenchymal cells in vitro is very similar.Flow cytometry assay showed that the expression rate of CD44 positive FITC was 98.34%,CD90 positive FITC expression rate was 99.14%,CD105 positive FITC expression rate was 99.97%,CD34 and CD45 negative expression rate were 1.05%and 0.08%,respectively.After immunofluorescence staining,the passage cells showed more cells in the field of vision with cell proliferation marker Ki67,indicating that most of the cells were in the division phase.2.In vitro differentiation and expression of Wnt7a in bone marrow mesenchymal stem cells of people.After osteogenesis,a large number of calcium deposits were produced,and the mRNA of OCN,OPN and Wnt7a increased significantly.The Wnt7a protein increased significantly.After the formation of fat,there is a lot of fat formation.The mRNA of PPARG gamma and LPL increased significantly.The expression of Wnt7a mRNA and protein decreased significantly.3.Effect of silencing and overexpression of Wnt7a gene on bone marrow mesenchymal stem cells.After the Wnt7a gene was silenced,the expression of Wnt7a mRNA and protein decreased significantly.After induction of osteogenic differentiation,no calcium nodules were generated,and the expression of OCN,OPN,Wnt7a,RUNX2 mRNA and protein decreased significantly.The decrease of transcription and expression of OSX was not significant.After overexpression of Wnt7a gene,the expression of Wnt7a mRNA and protein increased significantly.After induction of osteogenic differentiation,a large number of calcium nodules are generated.The expression of OCN,OPN,Wnt7a,RUNX2 mRNA and protein increased significantly.The decrease of transcription and expression of OSX was not significant.4.Wnt7a regulates bone differentiation in bone marrow mesenchymal stem cells through expression of RUNX2.Luciferase assay showed that the activity of TCF1 coding region and transcription factor binding activity of Wnt7a promoter was significantly lower than that of control group(P<0.05)after silencing Wnt7a bone marrow mesenchymal stem cells and inducing osteogenic differentiation 6 days later.For over 6 days after over expression of Wnt7a bone marrow mesenchymal stem cells and osteogenic differentiation,the binding activity of TCF1 coding region to transcription factor of Wnt7a promoter is much higher than that of control group(P<0.05).Using CHIP test,8K sequence fragment of downstream protein and RUNX2 gene on TCF1 locus on anchor anchor protein were detected,results showed that in the silence of Wnt7a bone marrow mesenchymal stem cells into osteogenic differentiation after 7 days,and the protein antibody TCF1 encoding region was significantly lower than that in control group,with statistical significance the difference(P<0.05),and the 8K sequence downstream of the two anchor protein detection volume is very low,the difference was not statistically significant(P>0.05).Conclusions1.bone marrow mesenchymal stem cells can be isolated and cultured in vitro by tissue block digestion and can be subcultured in vitro.In vitro,human bone marrow mesenchymal stem cells induced osteogenic differentiation,and the transcription and expression of Wnt7a gene increased significantly.When adipogenic differentiation was made,the transcription and expression of Wnt7a gene decreased significantly.2.Wnt7a gene silencing human bone marrow mesenchymal stem cells can cause osteogenic differentiation,but overexpression of Wnt7a gene can promote osteogenic differentiation,indicating that Wnt7a is involved in osteogenic differentiation of human bone marrow mesenchymal stem cells.3.The Wnt7a gene promotes the osteogenic differentiation of human bone marrow mesenchymal... |
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