RNA干扰热休克蛋白27基因对自发性高血压大鼠血管平滑肌细胞增殖、迁移的影响

Effects of HSP27 RNAi on Proliferation and Migration of Vascular Smooth Muscle Cells in Spontaneously Hypertensive Rats

目的研究自发性高血压大鼠热休克蛋白27(HSP27)基因的靶向小干扰RNA对血管平滑肌细胞增殖及迁移的影响。方法将血管平滑肌细胞(VSMC)随机分为对照组、血小板源生长因子(PDGF)组(10μg/L)、空载体组(pNL-EGFP)、干扰载体组(pNL-HSP27-EGFP)、干预组。以携带siRNA的慢病毒感染VSMC,通过细胞计数法和四甲基偶氮唑盐(MTT)比色法检测VSMC细胞增殖,使用PDGF诱导VSMC的HSP27磷酸化,用特异性的单克隆Phospho-HSP27抗体的蛋白免疫印迹法检测HSP27的活性,采用改良的Boyden微孔膜双槽法进行细胞迁移实验。结果 PDGF呈浓度依赖性诱导VSMC的HSP27磷酸化,与对照组相比增加168.9%(P<0.01);pNL-HSP27-EGFP对PDGF诱导的HSP27磷酸化的抑制率是79.6%(P<0.01)。pNL-HSP27-EGFP可显著抑制PDGF诱导的细胞增殖,抑制率为30.8%(P<0.05)。pNL-HSP27-EGFP能够显著抑制PDGF诱导的VSMC细胞迁移,抑制率为45.6%(P<0.01)。结论靶向HSP27基因的小干扰RNA能显著降低PDGF诱导VSMC细胞迁移,HSP27在细胞的迁移中起重要作用。

Objective To study the effect of lentiviral RNA interference （RNAi） sequence targeting the heat shock protein 27 （HSP27） gene of spontaneously hypertensive rats（SHR）on the proliferation and migration of SHR vascular smooth muscle cells （VSMCs）.Methods VSMCs infected by lentivirus were randomly divided into the control group,PDGF group,empty vector group,RNAi vector group,and intervention group.The VSMCs were transfected by shRNA lentivirus.Their proliferation was detected by daily count of cell number and methyl thiazolyl tetrazolium （MTT）.HSP27 phosphorylation was induced by PDGF.The activity of HSP27 was evaluated by Western blot with phospho-HSP27 antibody.Modified Boyden chamber technique was employed for VSMCs migration assessment.Results The proliferation of VSMCs induced by PDGF was significantly inhibited by pNL-HSP27-EGFP,with an inhibition rate of 30.8%（P0.05）.The phosphorylation of HSP27 in response to PDGF was suppressed by pNL-HSP27-EGFP,with a maximal inhibition rate of 79.6%（P0.01）.The migration of VSMCs induced by PDGF was significantly inhibited by pNL-HSP27-EGFP,with an inhibition rate of 45.6%（P0.01）.Conclusions The migration of VSMCs in response to PDGF is significantly inhibited by the RNAi targeting HSP27 gene.HSP27 may be a potential target for the interventional treatment of pathological process related to cell migration.