Smad7和uPA基因共表达对大鼠肝星状细胞生物学特性的影响  

Effects of Smad7 and uPA gene co-expression on biological characteristics of rat hepatic stellate cells

作　　者：宗春华[1] 汪保灿[1] 陈颖伟[1] 孙超[1] 李定国[1] 

机构地区：[1]上海交通大学医学院新华医院消化内科,上海200092

出　　处：《上海交通大学学报（医学版）》2007年第10期1173-1176,共4页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(30500236);上海市教委基金(05BZ49);上海市科委基金(064119527)~~

摘　　要：目的观察Smad7和尿激酶型纤溶酶原激活剂(uPA)基因共表达对体外培养肝星状细胞(HSC)活化、分泌胶原特性的影响。方法采用胶原酶原位灌注、密度梯度离心法分离大鼠HSC,将AdSmad7/uPA或AdSmad7体外感染HSC后,Western blot-ting法检测细胞总蛋白中Smad7、抗人α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原α2链(ColⅠA2)的表达;ELISA法检测培养上清中uPA蛋白的分泌量;放射免疫法测定HSC培养上清中Ⅲ型前胶原(PⅢP)和层黏连蛋白(LN)的分泌量。结果AdSmad7/uPA感染体外培养HSC,3 d后观察到绿色荧光蛋白(GFP)表达,转染效率>90%,Smad7和uPA蛋白的表达量明显高于AdGFP对照;AdSmad7/uPA或AdSmad7转染HSC均可使α-SMA、ColⅠA2、PⅢP和LN水平较AdGFP对照组显著下降(P<0.05)。与转染AdSmad7相比,转染AdSmad7/uPA后的HSC中ColⅠA2、PⅢP和LN表达降低更明显,分别较前者降低44.5%、30.4%和28.4%(P<0.05)。结论Smad7和uPA基因共表达可协同抑制体外培养的大鼠HSC分泌细胞外基质成分。Objective To investigate the therapeutic effects of Smad7 and urokinase-type plasminogen activator （uPA） co-expression on activation and collogen secretion of rat hepatic stellate cell （HSC） in vitro. Methods HSCs were isolated from male SD rats by in situ perfusion of liver and Nycodenz gradient centrifugation. After infection by AdSmad7/uPA or AdSmad7, the expressions of Smad7 and uPA in the HSCs were detected by Western blotting and enzyme linked immunosorbent assay （ELISA） , respectively. In addition, α- smooth muscle actin （α-SMA） and collagen Ⅰ α2 （Col Ⅰ A2） expressions were checked by Western blotting. The levels of extracellular matrix （ECM）, including procollagen Ⅲ peptide （PⅢ P） and laminin （LN） in the culture media were detected by radioimmunoassay methods. Results Three days after in vitro infection of HSC by AdSmad7/uPA, the expression of green fluorescent protein （GFP） was observed, the transfection efficiecy was more than 90% , and the expressions of Smad7 and uPA protein were significantly higher than the AdGFP controls. The expressions of α-SMA, Col Ⅰ A2, PⅢ P and LN were significantly decreased in HSCs transfected by AdSmad7/ uPA or AdSmad7 than in the AdGFP controls （P 〈0.05）. Compared with those transfected by AdSmad7, the expressions of Col Ⅰ A2, P Ⅲ P and LN were significantly decreased in HSCs transfected by AdSmad7/uPA, 44.5% , 30.4% and 28.4% lower than the former, respectively （P 〈0.05）. Conclusion Smad7 and uPA co-expression could synergistically reduce the contents of ECM in HSC.

关 键 词：SMAD7 尿激酶型纤溶酶原激活剂 腺病毒 基因治疗 肝星状细胞 

分 类 号：Q789[生物学—分子生物学]