导入外源人尿激酶型纤溶酶原激活剂基因对肝星状细胞胶原沉积的影响  

Effect of exogenous human urokinase-type plasminogen activator gene on the production of collagens in hepatic stellate cells

作　　者：林勇[1] 陈伟忠[1] 谢渭芬[1] 曾欣[1] 张新 陈岳祥[1] 张忠兵[1] 杨秀疆[1] 

机构地区：[1]第二军医大学附属长征医院消化内科,上海200003 [2]上海市国家人类基因组南方研究中心

出　　处：《中华消化杂志》2004年第7期387-390,共4页Chinese Journal of Digestion

基　　金：国家自然科学基金资助 ( 3 0 2 0 0 12 2 )

摘　　要：目的　利用重组复制缺陷型腺病毒载体将外源非分泌型人尿激酶型纤溶酶原激活剂(uPA)基因导入活化的肝星状细胞 (HSC)内 ,研究uPA表达对HSC胶原沉积的影响。方法　细菌内同源重组构建表达非分泌型uPA的复制缺陷型重组腺病毒AduPA。体外感染肝星状细胞株HSC T6 ,Northern印迹法和免疫细胞化学法检测uPA在HSC中的表达。酶联免疫吸附实验测定培养上清基质金属蛋白酶 2 (MMP 2 )分泌水平。免疫细胞荧光法测定外源uPA基因导入对HSC胞质内Ⅰ、Ⅲ型胶原含量变化的影响。结果　同源重组最终获得约 5× 1 0 1 1 efu/mlAduPA。AduPA体外感染HSC 3d后 ,Northern印迹法和免疫细胞化学法显示uPAmRNA及蛋白表达明显增加 ,细胞上清液中MMP 2分泌水平达 (2 74 .4 5± 7.6 3) pg/ml,明显高于对照组的 (1 4 5 .85± 6 .5 8)pg/ml(P <0 .0 1 )。免疫细胞荧光法提示uPA基因导入HSC后 ,胞质内Ⅰ、Ⅲ型胶原含量明显减少。结论　通过重组腺病毒载体将外源uPA基因导入HSC内可维持uPA高效表达并上调MMP 2分泌水平 ,减少细胞外基质沉积 。Objective To study the effect of exogenous non secreted human urokinase type plasminogen activator(uPA) gene on the production of collagen in activated hepatic stellate cells(HSC) after delivery by replication deficient recombinant adenovirus vector. Methods Non secreted human uPA was inserted into the replication deficient recombinant adenoviruses AduPA in bacteria. After infected by AduPA, the expression of uPA in HSC cell line HSC T6 was detected by Northern blot and immunocytochemical staining, and the level of matrix metalloproteinase 2 (MMP 2) in the supernatant of cultured HSC T6 cells was measured by ELISA. In addition, immunocytofluorescent staining was carried out to evaluate the effect of AduPA on the production of collagen Ⅰ and Ⅲ in HSC after exogenous uPA gene delivery. Results 5×10 11 efu/ml titer of AduPA was obtained in the HSC infected with the recombinated virus. Northern blot and immunocytochemical staining showed that the expression of uPA in HSC increased significantly on the third day after infection with the AduPA. The level of MMP 2 in the supernatant of the cultured HSC was (274.45±7.63) pg/ml, which was higher than that in the control [(145.85±6.58) pg/ml, P <0.01]. Additionally, the amount of collagen Ⅰ and Ⅲ in the cytoplasm of HSC decreased significantly after uPA gene delivery. Conclusions Our study revealed that uPA gene delivered into HSC by recombinant adenoviruses could be expressed with high efficiency in the cells, which is related to the up regulation of MMP 2 and attenuates the production of extracellular matrix in HSC. These results demonstrated that AduPA may serve as an effective way for gene therapy of hepatic fibrosis.

关 键 词：外源尿激酶型 纤溶酶原激活剂 基因 肝星状细胞胶原 胶原沉积 肝纤维化 

分 类 号：R575.2[医药卫生—消化系统]