Runx2重组慢病毒感染骨髓间充质干细胞并促进其成骨分化的研究

Research of lentiviral-mediated Runx2 transferred into bone marrow mesenchymal stem cells promoting osteogenic differentiation

目的利用重组Runx2基因的慢病毒感染大鼠骨髓间充质干细胞(BMSCs),使其Runx2基因的表达水平提高,观察BMSCs成骨相关基因的表达情况,研究Runx2基因促进BMSCs的成骨分化情况。方法 PCR扩增Runx2基因,并连接到慢病毒表达载体质粒Pez-lv201,与包装质粒共转染293T细胞进行包装,测得慢病毒液滴度。取4周龄SD大鼠胫骨,分离、培养BMSCs,流式细胞仪鉴定。将Runx2重组慢病毒感染BMSCs。显微镜下观察细胞形态变化;RT-PCR分析BMSCs成骨基因的表达情况。结果Runx2基因重组慢病毒表达载体质粒酶切和测序鉴定正确。测得慢病毒液滴度为1.6×109TU/ml。流式细胞仪检测表面抗原CD90和CD105,表达率为99.8%、99.3%。重组慢病毒感染后实验组细胞形态呈成骨样细胞改变;对照组未见明显变化。实验组Runx2、OCN、osteonectin、ALP、BMP-2、OPN基因的表达水平随时间推移而增高;对照组上述基因均无明显表达。结论利用Runx2重组慢病毒感染BMSCs,使其高表达Runx2基因,可以使OCN、osteonectin、ALP、BMP-2、OPN基因表达增强,说明Runx2基因可以促进BMSCs向成骨方向分化。

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 ＆#215; 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.