siRNA干扰明胶酶对人肾小管细胞ICAM-1表达的影响

Intervention of siRNA to the effect of gelatinase on the expression of ICAM-1 in human renal tubular epithelial cells

目的利用小干扰RNA(siRNA)抑制人肾小管上皮细胞(HKC)的基质金属蛋白酶-2(MMP-2)和MMP-9,即明胶酶A和B的表达,观察其对胞间黏附分子1(ICAM-1)表达的影响。方法培养人肾小管上皮细胞,以100nmol/L佛波醇酯(PMA)刺激18h,脂质体转染抑制MMP-2或MMP-9表达的siRNA或无关siRNA。提取细胞总RNA或胞浆蛋白,利用Real-TimePCR、Western blot或免疫荧光结合激光共聚焦显微镜技术检测MMP-2、MMP-9或ICAM-1的表达情况。结果特异性的siRNA能有效抑制HKC中MMP-2、MMP-9的mRNA和蛋白表达;免疫荧光结果显示经PMA刺激后HKC的ICAM-1表达上调,同时MMP-9-siRNA转染组人肾小管细胞的ICAM-1蛋白表达水比其他实验组高(P<0·05)。结论抑制肾小管上皮细胞MMP-9的表达可使ICAM-1表达上调,提示除了细胞外基质降解途径外,MMP-9还可通过炎症途径影响肾脏纤维化进程。

Objective To inhibit the expression of matrix metallopmteinase-2 （MMP-2） and matrix metalloproteinase-9 （MMP-9） in human kidney tubular epithelial cell （HKC） with specific small interference RNA （siRNA）, then to observe whether the expression of intercellular adhesion molecule-1 （ICAM-1） on HKC could be influenced. Methods HKCs were transfected with MMP-2, MMP-9 siRNA or irrespective siRNA respectively, untransfected like was considered as blank control. Four groups of HKCs were stimulated with 100nmol/L phorbol myristate acetate （PMA） for 18h. Total RNA or protein of cells were extracted with Trizol and RIPA cell lysate respectively, the mRNA expression levels of MMP-2, MMP-9 and ICAM-1 were detected by real time PCR, the protein expression levels of above indexes were detected by western blotting and immunofluorescence combined with confocal laser microscopy. Results The specific siRNA could inhibit the expression of MMP-2 and MMP-9 in HKCs effectively. Immunofluorescence combined with confocal assay demonstrated that while HKCs was stimulated with PMA, the expression of ICAM-1 was up-regulated, which was significantly up-regulated in the group transfected with MMP-9 siRNA （P〈0. 05）. However, there was no significant difference among the groups transfected with MMP-2 siRNA, irrespective siRNA and untransfected group （P〉0. 05）. Conclusions The inhibitory expression of MMP-9 of HKC may up-regulate the expression of ICAM-1, implying that, besides taking part in the degradation of extracellular matrix, MMP-9 may throw an influence on the proceeding of renal fibrosis by inflammatory pathway.