骨桥蛋白特异性短发夹RNA抑制大鼠血管平滑肌细胞增殖

Effect of Osteopontin-specific Small Hairpin RNA on Proliferation of Vascular Smooth Muscle Cells of Rats

目的应用特异性短发夹RNA(shRNA)抑制血管平滑肌细胞骨桥蛋白基因表达,观察其对体外培养的血管平滑肌细胞增殖的影响。方法根据大鼠骨桥蛋白基因设计靶序列,采用体外转录方法获取shRNA,以此转染血管平滑肌细胞,采用逆转录-聚合酶链反应、Western blot等方法检测骨桥蛋白mRNA和蛋白的表达;用MTT法检测shRNA对血管平滑肌细胞增殖的影响。结果与未转染特异性shRNA细胞相比,转染shRNA的细胞中骨桥蛋白mRNA、蛋白表达量均明显降低,抑制率分别为(76.3±3.4)%和(68.7±4.6)%。血管平滑肌细胞的增殖也受到明显的抑制。结论设计的靶序列能有效沉默血管平滑肌细胞骨桥蛋白基因的表达,为进一步构建载体和体内研究奠定了基础。

Objective To study the inhibitory effects of osteopontin(OPN)-specific small hairpin RNA(shRNA) on the expression of osteopontin(OPN) and its effect on the proliferation of in vitro cultured vascular smooth muscle cells in rats.Methods One target sequence was designed according to rats OPN sequence information from Dharmacon siRNA Design Center and shRNA was transcribed in vitro.The shRNA was transfected into vascular smooth muscle cells.Semi-quantitative polymerase chain reaction and Western blot were carried out to detect the expression of OPN mRNA and protein respectively.The cell proliferation was assayed by MTT.Results Compared with the blank control cells,the expression of OPN mRNA and protein in the cells transfected with shRNA was significantly decreased with the inhibition rate being(76.3±3.4)% and((68.7)±4.6)% respectively.The proliferation of vascular smooth muscle cells was inhibited obviously when OPN gene expression was knocked down by RNA interference(RNAi).Conclusion The expression of OPN gene can be efficiently inhibited in vitro by RNAi technique.These results are informative for further vector construction and study in vivo.