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目的:探讨ROCK亚型ROCKⅠ和ROCKⅡ对血管平滑肌细胞(A7r5)迁移及增殖的影响。方法:利用Western blot技术检测ROCKⅠ和ROCKⅡ蛋白在A7r5细胞中的表达水平;利用siRNA技术使ROCKⅠ和ROCKⅡ基因表达分别下调,并检测基因下调后蛋白表达水平;利用Boyden小室法,观察ROCKⅠ和ROCKⅡ基因下调后及ROCK特异抑制剂Y-27632对PDGF诱导的A7r5细胞迁移的影响;使用MTT法检测ROCKⅠ和ROCKⅡ基因下调后对A7r5细胞生长曲线的影响。结果:ROCKⅠ和ROCKⅡ在A7r5细胞中的蛋白表达水平不同,ROCKⅡ较ROCKⅠ的表达水平高4倍;通过对A7r5细胞进行ROCKⅠ和ROCKⅡsiRNA转染,使二者蛋白表达水平分别下调83.4%和94.7%;基因表达下调后,ROCKⅠ明显抑制了PDGF诱导的A7r5细胞的迁移,而ROCKⅡ无明显影响,Y-27632也抑制了A7r5细胞的迁移;ROCKⅠ和ROCKⅡ基因下调后对A7r5细胞生长曲线的影响无明显差别。结论:ROCKⅠ在血管平滑肌细胞迁移过程中起主导作用,ROCKⅠ和ROCKⅡ对血管平滑肌细胞的增殖作用无明显差异。
Objective: To investigate the effects of ROCK subtypes ROCK Ⅰ and ROCK Ⅱ on the migration and proliferation of vascular smooth muscle cells (A7r5). Methods: The expression of ROCKⅠ and ROCKⅡprotein in A7r5 cells was detected by Western blot. The expression of ROCKⅠ and ROCKⅡwere down-regulated by siRNA and the protein expression was detected by siRNA. The Boyden chamber method was used to observe the expression of ROCKⅠ and ROCKⅡ After down-regulation and ROCK-specific inhibitor Y-27632 on PDGF-induced A7r5 cell migration; using MTT assay ROCK Ⅰ and ROCK Ⅱ gene downregulation of A7r5 cell growth curve. Results: The protein expression levels of ROCK Ⅰ and ROCK Ⅱ in A7r5 cells were different. The expression level of ROCK Ⅱ was 4 times higher than that of ROCK Ⅰ. The expression of ROCK Ⅰ and ROCK Ⅱ siRNA were decreased by 83.4% and 94.7% respectively. ROCK Ⅰ significantly inhibited the migration of A7r5 cells induced by PDGF, while ROCKⅡ had no obvious effect, Y-27632 also inhibited the migration of A7r5 cells; The effect of ROCKⅠand ROCKⅡgene down-regulation on the growth curve of A7r5 cells showed no significant difference . Conclusion: ROCK Ⅰ plays a leading role in the migration of vascular smooth muscle cells. There is no significant difference in the proliferation of vascular smooth muscle cells between ROCK Ⅰ and ROCK Ⅱ.