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目的:检测点突变型HIF-1α对犬骨髓基质干细胞(bone marrow mesenchymal stem cells,BMSCs)骨向分化的作用。方法:采用LR重组系统构建慢病毒载体Lenti-WT(wild HIF-1α)、Lenti-MT(mutant HIF-1α)及对照组Lenti-LacZ。分别用Lenti-LacZ、Lenti-WT及Lenti-MT转染犬BMSCs,以LacZ组为对照,成功转染后,分别在0、1、4、7、14及21 d提取总RNA和蛋白质,通过RT-PCR和Western印迹检测目的基因在体外常氧条件下对BMSCs成骨因子表达的调控。将带有目的基因的BMSCs按2×105/孔接种至6孔板,分别于14 d和21 d,运用茜素红染色(alizarin red-S staining,ARS)检测其钙结节的表达。采用SPSS10.0软件包对数据进行统计学分析。结果:当MOI=9时,BMSCs的转染效率达90%以上。目的基因转染后第4天,BMSCs的成骨因子在mRNA和蛋白水平表达显著提高,14～21 d时达到高峰,并维持在高表达状态(P<0.05)。ARS结果表明,目的基因在常氧条件下可诱导BMSCs向成骨方向分化。结论:体外常氧条件下,突变型HIF-1α能够稳定表达且保持高度活性,Lenti-MT可显著提高犬BMSCs的成骨活性。
Objective: To investigate the effect of point mutation HIF-1α on bone differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods: Lenti-WT (Lenti-WT), Lenti-MT (mutant HIF-1α) and control group Lenti-LacZ were constructed by LR recombination system. Canine BMSCs were transfected with Lenti-LacZ, Lenti-WT and Lenti-MT respectively. LacZ group was used as control. After transfection, the total RNA and protein were extracted at 0, 1, 4, 7, 14 and 21 d RT-PCR and Western blotting were used to detect the regulation of osteogenic factor in BMSCs under normoxia. BMSCs with the target gene were inoculated into 6-well plates at 2 × 10 5 / well and the calcium nodules were detected by alizarin red-S staining (ARS) on day 14 and day 21, respectively. SPSS10.0 software package for statistical analysis of the data. Results: When MOI = 9, the transfection efficiency of BMSCs was over 90%. On day 4 after transfection, the osteogenic factors of BMSCs were significantly increased at mRNA and protein levels and peaked at 14-21 days (P <0.05). ARS results showed that the target gene can induce BMSCs to differentiate into osteoblasts under normoxia. CONCLUSION: In vitro hyperoxia, mutant HIF-1α can be stably expressed and highly active. Lenti-MT can significantly enhance the osteogenic activity of BMSCs.