金属蛋白酶组织抑制剂1基因转染对系膜细胞表达纤连蛋白和Ⅳ型胶原的影响

Effect of TIMP-1 on expression of FN and type Ⅳ collagen in rat glomerular mesangial cells

目的 探讨金属蛋白酶组织抑制剂(TIMP)-1对大鼠肾小球系膜细胞表达纤连蛋白(FN)和Ⅳ型胶原的影响。方法 应用亚克隆技术构建正义pLXIN-TIMP-1(PLT)和反义pLXIN-ATIMP-1(PLA)两个逆转录病毒载体,经PA317细胞包装后,得到高滴度的病毒上清,分别感染大鼠系膜细胞,用PCR及Northern杂交鉴定外源基因的整合和表达。Northern杂交及ELISA方法检测大鼠系膜细胞内源性TIMP-1、FN和Ⅳ型胶原的表达。结果 外源TIMP-1基因稳定整合到大鼠肾小球系膜细胞中,并获得高效表达。感染正义TIMP-1基因后,大鼠系膜细胞内源性TIMP-1表达没有变化,细胞外基质成分FN和Ⅳ型胶原的蛋白质水平的表达明显增高;与之相反,感染反义的TIMF-1基因后,大鼠内源性TIMP-1下调,细胞外基质成分FN和Ⅳ型胶原的蛋白质水平的表达明显降低。然而,上述系膜细胞FN和Ⅳ型胶原的mRNA水平均没有变化。结论TIMP-1过表达引起FN和Ⅳ型胶原增多,反义TIMP-1则使FN和Ⅳ型胶原的表达下调。提示反义TIMP-1可通过促进肾脏细胞外基质降解而减轻其积聚,为进一步在某些纤维化肾脏疾病中应用反义TIMP-1进行基因治疗奠定一定的实验基础。

ObjectⅣe To investigate the effect of sense and antisense TIMP-1 on the expression of FN and type Ⅳ collagen of rat glomerular mesangial cells in vitro. Methods Two recombinant retroviral vectors, pLXIN-TIMP-1 (PLT)encoding sense TIMP-1 and pLXIN-ATIMP-1 (PLA)encoding antisense TIMP-1 were constructed by using DNA recombining techniques. These two vectors were then introduced into the PA317 packaging cell line with lipofectin DOTAP. The high-titer retroviral supematants containing sense or antisense TIMP-1 were used to infect rat glomerular mesangial cells. PCR and Northern blot were used to detect the integration and expression of human TIMP-1. Northern blot and ELISA were employed to investigate the expression of endogenous TIMP-1, FN, and Ⅳ collagen. Results Both sense and antisense TIMP-1 were successfully integrated into rat mesangial cells and expressed the sense and antisense ITMP-1 RNA. Overexpression of TIMP-1 induced by sense TIMP-1 caused upregulation of FN and Ⅳ type collagen in protein level, in contrast, supprssion of TIMP-1 expression by antisense TIMP-1 caused downregulation of FN and Ⅳ type collagen protein; but neither sense nor antisense TIMP-1 infection had effects on the RNA level of FN and Ⅳ type collagen. Conclusion TIMP-1 suppresses the degradation of ECM in rat glomerular mesangial cells and antisense TIMP-1 may be available for renal fibrosis in the future.