摘要
目的 通过大鼠肾小球系膜细胞 (mesangialcell,MsC)转染Smad7基因 ,观察转基因MsCMMP 2、uPA和ColⅣ表达的改变 ,以阐明Smad7在肾小球硬化发生中的作用及其机制。方法 经脂质体介导将含有Smad7重组表达质粒转染大鼠MsC ,用G4 18筛选及Westernblot、RT PCR法鉴定 ;采用Westernblot、酶谱分析和RT PCR法 ,检测转染阳性MsC克隆MMP 2、uPA和ColⅣ表达改变。结果 成功建立高表达Smad7的阳性MsC克隆 (S 2 2 ,S 2 6 ) ,并证实其MMP 2蛋白分泌和酶活性明显增加 ,以及uPAmRNA及其蛋白的表达明显增强 ,并伴有MsCColⅣ合成的显著下降。结论 Smad7可能通过增强MsCMMP 2。
Purpose To elucidate the mechanism of Smad7 antagonizing glomerulosclerosis by observing matrix metalloproteinase 2 (MMP 2), urokinase type plasminogen activator (uPA) and type Ⅳ collagen expressions on cultured rat mesangial cells(MsC) transfected with Smad7 vector. Methods Lipofectin method was used to transfect Smad7 vector into rat MsC, Western blot and RT PCR analysis for detecting Smad7 protein and mRNA expression level. The expressions of MMP 2, uPA and typeⅣ collagen were determined by Western blot, RT PCR and Zymography assay,respectively. Results Overexpresion of Smad7 on two positive MsC clones (S 22,S 26) were successfully established. Two MsC clones showed increased expression of MMP 2 protein , enhanced enzyme activity and increased expressions of uPA protein and its mRNA,accompanied with decreased synthesis of type Ⅳ collagen protein. Conclusions It is possible that Smad7 can alleviate the development of glomerulosclerosis by upregulating the expression of MMP 2 and uPA accompanied with decreased synthesis of type Ⅳ collagen.
出处
《临床与实验病理学杂志》
CAS
CSCD
2004年第1期83-86,共4页
Chinese Journal of Clinical and Experimental Pathology
基金
上海科技发展基金资助项目 (No 0 1JC14 0 18)
