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

发布时间：2018-03-08 01:31

本文选题：基质金属蛋白酶-　切入点：血管平滑肌细胞　出处：《中国循环杂志》2015年02期 　论文类型：期刊论文

【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%(P0.05),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。
[Abstract]:Objective: to study the effect of small interfering ribonucleic acid siRNAs targeting matrix metalloproteinase-3 gene on proliferation of vascular smooth muscle cells in rats. Methods: three pairs of nuclei targeting matrix metalloproteinase-3 (MMP-3) in spontaneously hypertensive rats were constructed. The vascular smooth muscle cells cultured in vitro were randomly divided into control group (Lipofectamine 2000) group, MMP-3-si RNA-1 group, MMP-3-si RNA-2 group and MMP-3-si RNA-3 group. After transfection of vascular smooth muscle cells with Lipofectamine 2000 for 48 h, the expression of MMP-3 mRNA mRNA in vascular smooth muscle cells was detected by real-time quantitative polymerase chain reaction (PCR). Si RNA, which has the highest interference efficiency, was selected as the experimental object. Vascular smooth muscle cells were randomly divided into four groups: control group, platelet derived growth factor (PDGF) group, Lipofectamine 2000 PDGF group and MMP-3-si RNA-1 PDGF group, the vascular smooth muscle cells were transfected with si RNA in vitro. The proliferation of vascular smooth muscle cells was detected by MTT colorimetry. Results: Real-time quantitative PCR showed that three pairs of complementary interference fragments had different degree of interference effects. Compared with the control group, the level of m RNA in MMP-3-si RNA-1 group and MMP-3-si RNA-3 group was significantly lower than that in MMP-3-si RNA-2 group. The interference efficiency of MMP-3-si RNA-1 group was the highest. Western blot analysis also showed that MMP-3-si RNA-1 group had the highest interference efficiency. Compared with PDGF group, the transfection of MMP-3-si RNA-1 PDGF could significantly decrease the absorbance of PDGF induced proliferation of vascular smooth muscle cells, and the inhibitory rate was 36.1% (P 0.05). Conclusion: the target MMP-3 gene si RNA sequence is significantly different from that of MMP 3-si RNA-1 PDGF group. It can significantly reduce the level of MMP-3 m RNA and protein and inhibit the proliferation of smooth muscle cells. The interference of MMP-3 gene with RNA may become a new target for the treatment of vascular proliferative diseases.
【作者单位】：郑州大学附属郑州中心医院心血管内科;
【基金】：郑州市科技攻关计划项目(No:112PPTSF312-3)
【分类号】：R965

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