核糖核酸干扰基质金属蛋白酶-3基因对大鼠血管平滑肌细胞增殖的影响  

Effect of RNA Small Interfering Sequence of MMP-3 on Vascular Smooth Muscle Cell Proliferation in Experimental Rats

作　　者：黄捷[1] 冯艳[1] 韩凌[1] 李艳[1] 张晶晶[1] 宋昆鹏[1] 石海莉[1] 

机构地区：[1]郑州大学附属郑州中心医院心血管内科,河南省郑州市450007

出　　处：《中国循环杂志》2015年第2期159-163,共5页Chinese Circulation Journal

基　　金：郑州市科技攻关计划项目(No:112PPTSF312-3)

摘　　要：目的:研究靶向大鼠基质金属蛋白酶-3基因的小干扰核糖核酸(si RNA)对血管平滑肌细胞增殖的影响。方法:构建靶向自发性高血压大鼠基质金属蛋白酶-3(MMP-3)的三对核糖核酸(RNA)干扰序列。将体外培养的血管平滑肌细胞随机分为对照组、Lipofectamine 2000(转染试剂)组、MMP-3-si RNA-1组、MMP-3-si RNA-2组、MMP-3-si RNA-3组,以Lipofectamine 2000分别转染血管平滑肌细胞,48 h后,以实时定量聚合酶链反应(PCR)检测血管平滑肌细胞MMP-3信使核糖核酸(m RNA)的表达水平,筛选干扰效率最高的si RNA作为实验对象。血管平滑肌细胞随机分为4组:对照组、血小板源生长因子(PDGF)组、Lipofectamine 2000+PDGF组及MMP-3-si RNA-1+PDGF组;用si RNA转染体外培养的血管平滑肌细胞,以四甲基偶氮唑盐(MTT)比色法检测血管平滑肌细胞增殖。结果:实时定量PCR显示,三对互补干扰片段均有不同程度的干扰效果,与对照组比较,MMP-3-si RNA-1组、MMP-3-si RNA-2组和MMP-3-si RNA-3组的m RNA水平明显降低,分别降低了81%、64%和22%。以MMP-3-si RNA-1组的干扰效率最高。蛋白免疫印迹法分析结果亦显示MMP-3-si RNA-1组的干扰效率最高。四甲基偶氮唑盐比色法检测吸光度值结果显示,与PDGF组相比,MMP-3-si RNA-1+PDGF组转染能显著降低PDGF诱导血管平滑肌细胞增殖的吸光度值,抑制率为36.1%(P<0.05),差异有统计学意义。结论:靶向MMP-3基因si RNA序列,能显著降低平滑肌细胞MMP-3 m RNA和蛋白水平,抑制细胞增殖。靶向MMP-3基因干扰RNA可能成为血管增殖性疾病治疗的新靶点。Objective： To study the effect of RNA small interfering （siRNA） sequence of matrix metalloproteinase-3 （MMP-3） on vascular smooth muscle cell （VSMC） proliferation in experimental rats. Methods：There were 3 complementary targeting sequences of MMP-3 gene were established according to MMP-3 gene sequence in Genbank as MMP-3-siRNA-1, MMP-3-siRNA-2 and MMP-3-siRNA-3. The VSMCs were cultured in 5 groups：①Control group,②Lipofectamine2000 group,③MMP-3-siRNA-1 group,④MMP-3-siRNA-2 group and⑤MMP-3-siRNA-3 group. The cells were transfected with Lipofectamine2000 for 48 hours and the mRNA expression of MMP-3 was examined by quantitative RT-PCR （qRT-PCR）. The siRNA sequence with the highest interfering efifciency was selected for further experiment, and VSMCs were divided in 4 groups：①Control group,②PDGF group,③Lipofectamine2000＋PDGF group and④MMP-3-siRNA-1＋PDGF group. The cells were transfected with siRNA, and the proliferation of VSMC was detected by MTT method. Results： qRT-PCR presented that 3 complementary sequences had different degrees of interfering efifciency. Compared with Control group, the mRNA expressions of MMP-3 decreased in MMP-3-siRNA-1 group, MMP-3-siRNA-2 group and MMP-3-siRNA-3 group at 81%, 64%and 22%respectively. The MMP-3-siRNA-1 sequence had the highest interfering efifciency and the same result was obtained by western blot analysis. Compared with PDGF group, MMP-3-siRNA-1＋PDGF group showed signiifcantly decreased VSMC proliferation by 36.1%, P〈0.05. Conclusion： The siRNA targeting sequence may decrease the mRNA and protein expression of MMP-3 and inhibit VSMC proliferation in experimental rats; it may become a new targeting point for treating the relevant vascular diseases.

关 键 词：基质金属蛋白酶-3 血管平滑肌细胞 RNA干扰 增殖 

分 类 号：R541[医药卫生—心血管疾病]