| Objective:This study aims to research the influence of Wnt10b on human bone marrow mesenchymal stem cells (BMSCs) when it is high expression or loss of expression by using Wnt10b gene expression adenoviral vector and Wnt10b-shRNA adenoviral vector, and explore the molecular mechanism of how Wnt10b regulate their differentiation into osteoblast or lipocyte through the canonical Wnt signaling pathway, which can provide experimental support and theoretical basis for gene therapy of osteoporosis.Methods:Clinical collecting the bone marrow from patients who underwent the total hip replacement and isolating the bone marrow mononuclear cells by using the percoll gradient centrifugation, which could get the human BMSCs, and then purified the human BMSCs according to the growth characteristics of adherent cells, after that, the human BMSCs could be subcultured and identified by the flow cytometry, finally,2-4generations were selected for osteogenic and adipogenic differentiation, ALP staining, Alizarin Red S staining and Oil Red-O staining were used for detecting. The adenovirus vectors (Ad-WntlOb, Ad-WntlOb-shRNA, Ad-Null) were constructed and identified first, after that, they were transiently transfected into human BMSCs which comprised four groups:Wnt10b group (Ad-Wnt10b), Wnt10b interference group (Ad-WntlOb-shRNA), empty virus group (Ad-Null) and control group, and the transfection efficiency was detected by immunofluorescence, then the transfected cells were transferred and cultured in inducing medium (osteogenic culture, adipogenic culture or normal culture), mRNA and protein of specific functional genes (ALP) and key regulatory transcription factor (Runx2) of osteogenic differentiation and specific functional genes (FABP4) and key regulatory transcription factor (PPARy2) of adipogenic differentiation (FABP4) were detected by fluorescence quantitative PCR, ALP staining, immunofluorescence, Western blot, alizarin red staining and oil red staining, finally, the influence of overexpression or inhibition of Wnt10b on osteogenic and adipogenic differentiation was comprehensive analysed.Results:The bone marrow mononuclear cells were successfully got by using the percoll gradient centrifugation and purified according to the growth characteristics of adherent cells, and cells were successfully identified, subcultured and differentiated into osteoblast or lipocyte in the inducing medium. The adenovirus vectors (Ad-Wnt10b, Ad-Wnt10b-shRNA, Ad-Null) were successfully constructed and transfected into human BMSCs,6h after transfection, the high transfection efficiency was detected by immunofluorescence.3d after induction, quantitative PCR showed: ALP and Runx2of osteogenic differentiation and FABP4and PPARy2of adipogenic differentiation were significantly higher than that of normal group, the expression of osteogenic factors associated with Wnt10b group were higher than that of other control group and the expression of adipogenic factors lower than that of other control group, but the Wnt10b interference group had the opposite results.7d after induction, ALP staining of WntlOb group in osteogenic culture was strongly positive, and the quantitative ALP was higher than that of control group, while in Wnt10b interference group, it was weakly positive, and the quantitative ALP was lower than the control group. Immunofluorescence and Western Blot analysis showed:WntlOb group in osteogenic culture had a higher expression of ALP and Runx2, while they were lower than the control group in Wnt10b interference group, and Wnt10b group in adipogenic culture had a lower expression of FABP4and PPARy than that of control group, while the Wnt10b interference group was higher than other groups.14d after induction, Oil Red-0staining of Wnt10b interference group in adipogenic culture showed strongly positive, while Wnt10b group was weakly positive,21d after induction, Red S staining of WntlOb group in osteogenic culture was strongly positive, while Wnt10b interference group was weakly positive.Conclusion:Human BMSCs were successfully isolated, cultured and identified, and they could be differentiated into osteoblast or lipocyte in the inducing medium, Overexpression of Wnt10b gene could promote the osteogenic differentiation of human BMSCs, while inhibiting their adipogenic differentiation, but inhibition the expression of Wnt10b gene, it showed the opposite results. |
