microRNA-21对体外培养血管平滑肌细胞迁移能力的调控  

Effect of microRNA-21 on migration and invasion of rat vascular smooth muscle cell in vitro

作　　者：李宪伟[1] 姜维良[1] 张英男[1] 孙占峰[1] 孙庆峰[1] 张天华[1] 马军[1] 

机构地区：[1]哈尔滨医科大学附属第二医院血管外科,哈尔滨150086

出　　处：《中国血管外科杂志（电子版）》2010年第4期213-218,共6页Chinese Journal of Vascular Surgery(Electronic Version)

摘　　要：目的探讨调控体外培养大鼠血管平滑肌中微小RNA-21(microRNA-21,miRNA-21)的表达水平对该类细胞迁移和侵袭能力的影响。方法 0.1%Ⅱ型胶原酶消化分离血管平滑肌细胞并进行培养,用免疫组化染色法检测细胞胞浆内α-肌动蛋白的表达。试验分为6组:空白对照组(加DEPC水)、单纯转染试剂组(仅加lipofectamineTM2000)、阴性对照组(转染阴性对照序列)、上调miRNA-21组(转染miRNA-21拟似物)、下调miRNA-21组(转染miRNA-21阻遏物)、无关序列组(转染无关序列)。利用lipofectamineTM2000将miRNA-21转染到培养的血管平滑肌细胞中,采用real-timePCR分别检测各组细胞miRNA-21的表达水平。采用细胞划痕实验分别检测各组细胞体外迁移能力的变化和transwell侵袭小室模型分别检测各组细胞侵袭能力的变化。结果原代血管平滑肌细胞在相差显微镜下呈现‘谷和峰’的生长特点,胞浆内α-肌动蛋白染色阳性的细胞数占总细胞数的98%以上。与对照组相比,上调miRNA-21组的血管平滑肌细胞体外迁移和侵袭能力明显增强(P<0.05),下调miRNA-21组的血管平滑肌细胞体外迁移和侵袭能力明显降低(P<0.05)。结论 miRNA-21可以促进血管平滑肌细胞迁移和侵袭。Objective To establish a method for culture of rat vascular smooth muscle cell(VSMC) in vitro, then to investigate the effect of microRNA-21(miRNA-21) on the migration and invasion of VSMC. Methods 0.1% collagenase Ⅱ was used to digest and isolate the thoracic aorta VSMC of rat and then cultured. And expression of α-actin of cytoplasm of VSMC was analyzed with immunocytochemistry. Then VSMCs were grouped as follows: blank control grpup (only DEPC), transfection reagent group (lipofectamineTM2000), negative control group (transfection of negative control gene sequence) , up-regulation of miRNA-21 group (transfection of miRNA-21mimics),down-regulation of miRNA-21 group(transfection of check of miRNA-21 ), unrelated sequence group (transfection of unrelated sequence). Synthesized miRNA-21 was transfected with lipofectamineTM2000 into cultured rat aortic VSMC in vitro, and the expression of miRNA-21 of thses 6 groups was detected by real-time PCR, respectively. Migration ability of VSMC of thses 6 groups was measured by scratch wound model and invasion ability by Transwell chamber model in vitro. Results The cultured cells exhibited the characteristic 'hills and valleys' growth pattern as observed by phase contrast microscopy and over 98%α-actin of cytoplasm stained. In up-regulation of miRNA-21 group, overexpression of miRNA-21in VSMCs increased cell migration and invasiveness(P<0.05), whereas in down-regulation of miRNA-21 group, inhibition of miRNA-21 in VSMCs reduced cell migration and invasiveness(P<0.05)compared with other control groups. Conclusion miRNA-21 can effectively promote the migration and invasion of the VSMC.

关 键 词：血管平滑肌细胞 微小RNA-21 迁移 侵袭 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]