【摘要】 体外构建及筛选小鼠Smad6重组慢病毒干扰载体,并在骨髓间充质干细胞(BMSCs)中检测其干扰效果。通过分子克隆技术应用携带绿色荧光蛋白(GFP)的慢病毒构建三个重组Smad6 RNA干扰载体,并通过DNA测序确定基因重组的正确性。培养小鼠BMSCs,并用骨形态发生蛋白2(BMP2)进行骨向诱导。应用激光共聚焦技术确定重组病毒的转染效率,并通过Real-time PCR和Western blot对Smad6基因的干扰效果进行检测。实验成功构建了三个Smad6重组慢病毒干扰载体,DNA测序证实了基因重组的正确性。重组病毒转染间充质干细胞(MSCs)后,GFP均得到了有效表达,达到了较高的转染效率(>95%)。经Real-time PCR和Western bloot检测,#2重组载体对Smad6基因表达的干扰效果最佳,蛋白水平干扰效率接近91%。本实验成功构建了小鼠Smad6基因有效重组干扰载体,为BMP信号通路的进一步研究提供了有效的实验工具。更多还原

【Abstract】 This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells(BMSCs).Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein(GFP),and the correctness of recombinant vectors was verified by DNA sequenceing.Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs.The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot,respectively.Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing.After transfection,GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency(>95%).Both real-time PCR and Western blot examination indicated that among three recombinant vectors,No.2 Svector had the best interference effect and the interference effect was nearly 91% at protein level.In conclusion,Mouse recombinant Smad6 RNA interference(RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.更多还原