Research of lentiviral-mediated Runx2 transferred into bone marrow mesenchymal stem cells promoting osteogenic differentiation
Chinese Journal of Clinical Anatomy ›› 2015, Vol. 33 ›› Issue (3) : 311-315.
Research of lentiviral-mediated Runx2 transferred into bone marrow mesenchymal stem cells promoting osteogenic differentiation
Objective To obtain the recombinant BMSCs with the reprogramming Runx2 gene mediated by lentiviral vectors, and detect the levels of the osteogenesis-related genes expression, in order to elucidate that Runx2 is able to promote BMSCs' differentiation. Methods Runx2 genes were amplified by RT-PCR and linked to the lentivirus vector, then transfected 293T cells together with packing plasmids. Lentivirus titer was identified. BMSCs were obtained from 4 weeks old SD rats' tibia bone marrow and isolated and were cultured. Analysis of cell surface markers CD90 and CD105 was performed by flow cytometry to confirm the cultured cells were BMSCs, which were finally transfected by Runx2 recombinant lentivirus. The expression of the osteogenic-related gene was studied through RT-PCR. Results Runx2 gene linked with expression vector, was confirmed through enzyme digestion and sequencing. The virus tilter was 1.6×109 TU/ml. The expression percentage of CD90 and CD105 was 99.8%and 99.3%, indicating the successful culture of BMSCs. After being infected with lentivirus, cells of BMSCs-Runx2 became osteogenous. The control BMSCs stayed the same as pre-infected. The expression of Runx2, OCN, osteoectin, ALP, BMP-2, OPN increased over time and achieved the highest on day 14 in experimental groups, while the control group didn't show the expression, verifying that Runx2 is able to promote BMSCs' differentiation.Conclusion Lentiviral-mediated Runx2, which was transfered into bone marrow mesenchymal stem cells, could promote the osteogenic differentiation.
Tissue engineering / Runx2 / Bone mesenchymal stem cells / Lentivirus
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