RNA干扰抑制c-myc基因对体外培养大鼠血管平滑肌细胞活性的影响

RNA interfering to c-myc increases the activity of cultured rat vascular smooth muscle cells in vitro

目的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖是导致移植静脉再狭窄的主要原因之一,早期应答基因c-myc是促进平滑肌细胞持续增殖的关键基因。探讨针对c-myc基因的RNA干扰对体外培养平滑肌细胞增殖和活性的影响。方法体外培养大鼠VSMCs,构建3对针对c-myc基因的RNA干扰序列(siRNA-1#、siRNA-2#、siRNA-3#)和1组阴性对照序列,以Lipo2000为载体转染体外培养细胞,Q-RT-PCR检测c-myc mRNA表达,Western blot检测转染c-myc蛋白表达,MTT法检测细胞增殖状态和活性。结果转染3对siRNA序列48h后,各组c-myc mRNA表达水平显著低于阴性对照组和正常细胞组,3组转染组之间c-myc mRNA表达无显著差异。转染siRNA-1#、siRNA-2#序列后,c-myc蛋白表达低于转染siRNA-3#序列组、阴性对照组和正常细胞组,而转染siRNA-3#序列组c-myc蛋白表达与阴性对照组和正常细胞组相比无明显差异。转染24 h各组间细胞活性未见显著差异,48h时细胞活性与24h相比无显著增加,至72h时各组细胞活性有明显增加,以转染siRNA-1#序列组增加量最少。阴性对照组和正常细胞组抑制效率基本保持为零水平。转染siRNA-1#、siRNA-2#序列后,24、48、72 h后抑制效率逐渐增加,转染siRNA-3#序列后72h后其抑制效率开始降低,各相同时间点以转染siRNA-1#序列后抑制效率最高。结论体外转染c-myc基因siRNA序列,能显著降低平滑肌细胞c-myc基因mRNA和蛋白表达,抑制细胞活性和增殖。随着作用时间延长,RNA干扰(RNA interference,RNAi)后细胞活性缓慢增加,而抑制效应持续显著增加,以siRNA-1#序列抑制作用最为明显。

Objective The proliferation of vascular smooth muscle cells（VSMCs） is one of the main causes of vein graft restenosis.It has been identified cmy-c,an immediate-early response gene,plays a key role in the continuous proliferation of VSMCs.The present study aims to explore the effect of small interfering RNA（siRNA） to c-myc on the proliferation and activity of cultured rat VSMCs in vitro.Methods We cultured rat VSMCs in vitro and synthesized three groups of RNA interfering sequences targeting the c-myc gene（siRNA-1#,siRNA-2#,siRNA-3#） and a group of scrambled siRNA.Then we transfected the cultured cells by Lipo2000,and determined the mRNA and protein expressions of c-myc by Q-RT-PCR and Western blot,respectively.The activity and proliferation were examined by means of MMT technique.Results At 48 hours after transfection,the expression of c-myc mRNA was significantly lower in the three transfected groups than in the negative and normal groups,with no significant differences among the former three,and so was the expression of c-myc protein in the cells transfected with siRNA-1# and siRNA-2# sequences than in the other three groups,with no significant differences between the siRNA-3# group and the negative and normal groups.The cell activity showed no remarkable differences among different groups at 24 hours,remained unchanged at 48 hours,but increased markedly at 72 hours,with the least elevation in the siRNA-1# group.The inhibition rate remained basically at zero in the negative and normal groups,increased gradually at 24,48 and 72 hours after tranfected with siRNA-1# and siRNA-2# sequences,and began to decline at 72 hours in the siRNA-3# group.The highest inhibition rate was observed in the siRNA-1# group at the same time point.Conclusion In vitro transfection of VSMCs with siRNA sequences targeting c-myc can significantly decrease the mRNA and protein expressions of c-myc and markedly inhibit the cell activity.With the prolonged time of trasfection,the cell activity is gradually increased,and the inhibitory effect continuously enhanced,most obviously for the siRNA-1# sequence.