点突变型HIF-1α诱导犬骨髓基质干细胞骨向分化的研究

The study of osteogenic differentiation in canine bone marrow stromal cell induced by point mutant type of HIF-1α

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。

PURPOSE： To evaluate osteogenesis in canine bone marrow mesenchymal stem cells（BMSCs） transduced by point mutant HIF-1α.METHODS： Lenti-WT（wild type HIF-1α）,Lenti-MT（mutant type HIF-1α）,and Lenti-LacZ（the control group） were constructed with LR recombination system.And then Lenti-LacZ,Lenti-WT and Lenti-MT were used to transduce canine BMSCs.After transduction,total RNA and protein were extracted at 0,1,4,7,14 d and 21 d, respectively.The mRNA and protein expression of osteogenic factors were detected by RT-PCR and Western blot in vitro under normoxic conditions.To further prove osteogenic differentiation of HIF-1α mediated BMSCs,the BMSCs were inoculated into 6-well plate（2 ×10 5 /well） and then the expression of calcium nodules was measured using Alizarin red staining（ARS） at 14 d and 21 d,respectively.RESULTS： At MOI = 9,the transduced efficiency of BMSCs was up to 90%.After target gene being transduced to BMSCs,the mRNA and protein expression of osteogenic factors were significantly up-regulated at 4d,reached peak at 14-21 d and maintained a state of high expression（P0.05）.ARS results showed that the target gene can induce BMSCs to the osteogenic differentiation in normoxic conditions.CONCLUSIONS： Under normoxic conditions in vitro,mutant HIF-1α can stabilize the expression and maintain a high level of activity.LentiMT can significantly improve the osteogenic activity of canine BMSCs.