腺病毒介导人uPA感染大鼠间充质干细胞的体外研究  

In vitro study of transfection of mesenchymal stem cells with adenoviral vector overexpressing human uPA

作　　者：胡俊杰[1] 孙超[1] 兰玲[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）》2009年第4期365-369,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(30500236);上海市科委基金(064119527);上海市教委优秀青年教师科研专项基金(18040)~~

摘　　要：目的体外分选与鉴定大鼠骨髓间充质干细胞(MSCs),并用腺病毒介导的人尿激酶型纤溶酶原激活物(uPA)体外感染原代MSCs,观察病毒的转导效率及对MSCs增殖的影响。方法通过黏附培养分离纯化MSCs,并采用免疫组化DAB显色法鉴定;荧光显微镜检测uPA的转染效率;Western blotting检测uPA在MSCs中的表达;MTT法检测病毒对MSCs增殖的影响。结果分选培养的MSCs呈典型的间充质干细胞生长形态,细胞表面标记物CD29和CD90阳性,CD34和CD45阴性;随着病毒感染滴度增高,绿色荧光蛋白(GFP)的表达率亦呈增高趋势。当感染复数(MOI)=80且病毒转导72 h后,GFP阳性细胞数占细胞总数的比例达(94.0±1.5)%,可稳定表达7 d以上,且对MSCs的增殖无明显影响。结论MSCs是一种理想的基因过表达载体,稳定分泌uPA蛋白,可用于肝纤维化的研究。Objective To identify the isolated rat bone marrow derived mesenchymal stem cells （MSCs）, and evaluate the efficiency of adenoviral vector expressing human urokinase type plasminogen activator （uPA） in transfection of rat MSCs and its effect on proliferation of MSCs. Methods MSCs were isolated and purified by pasted wall purification, and were identified by immunicytochemistry. The transfection efficiency of uPA was detected by fluorescent microscopy, the expression of uPA in MSCs was detected by Western blotting, and the proliferation of MSCs was evaluated by MTT. Results The harvested MSCs exhibited the typical appearance of MSCs, and it was revealed by immunohistoehemistry that the expression of MSCs markers CD29 and CD90 was positive, while that of CD34 and CD45 was negative. A tendency of increase in expression of green fluorescent protein （GFP） was observed with increase of multiplicity of infection （MOI）. After transfection with AduPA for 72 h, the transfection efficiency reached （94.0 ± 1.5） % at MOI of 80, and positive GFP cells could still be observed even after 7 d. The transfected uPA had no effect on the proliferation of MSCs. Conclusion MSCs are favourable genetic vectors to express uPA, and can be used for treatment of liver fibrosis.

关 键 词：间充质干细胞 尿激酶型纤溶酶原激活物 肝纤维化 

分 类 号：R373[医药卫生—病原生物学]