人Arresten基因的真核表达及其对血管平滑肌细胞增殖和迁移的影响  

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

作　　者：尚丹[1] 郑启昌[1] 宋自芳[1] 汪谢丹[1] 胡青钢[1] 郭兴军[1] 

机构地区：[1]华中科技大学同济医学院协和医院普外科,湖北武汉430022

出　　处：《中国病理生理杂志》2007年第10期1887-1890,共4页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30371396No.30271242)

摘　　要：目的:真核表达人Arresten蛋白,并研究其对体外培养大鼠胸主动脉血管平滑肌细胞(VSMCs)增殖和迁移的影响。方法:用脂质体介导,将含有人Arresten基因的重组质粒pSecTag2-AT转染COS-7细胞;应用逆转录-聚合酶链反应(RT-PCR)检测转染细胞目的基因mRNA表达;收集转染48h后上清液,浓缩后Westernblotting分析检测目的蛋白的表达。用转染细胞上清液处理平滑肌细胞后,CCK-8法检测细胞增殖;Transwell小室法检测细胞迁移。结果:RT-PCR结果显示转染Arresten基因的COS-7细胞基因组中存在有目的基因特异性片段(449 bp),表明基因转染成功;Western blotting结果表明转染细胞上清液中有目的蛋白表达。细胞体外增殖分析显示Arresten蛋白可显著抑制血管平滑肌细胞的增殖作用(F=40.154,P<0.01);Transwell小室法检测显示经对照细胞,载体DNA转染及重组质粒转染的COS-7细胞培养上清液处理的平滑肌细胞迁移数为(28.70±3.97)个、(26.10±4.53)个、(14.00±3.33)个(F=38.915,P<0.01)。结论:真核表达的人Arresten蛋白能有效抑制体外培养的血管平滑肌细胞的增殖和迁移。AIM： To express human Arresten gene in eukaryotic cell, and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells （VSMCs）. METHODS： COS -7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2 - AT or control plasmid pSecTa2 mediated by liposome. 48 hours after transfection, polymerase chain reaction （RT -PCR） was used to detect the expression of Arresten mRNA in the cells, while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants. VSMCs were then co - cultured with the concentrated supernatants; and its proliferation was detected using cell counting kit - 8 （ CCK - 8） in vitro. Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter （Transwell＇s chamber） with pores of 8 μm in diameter. RESULTS： RT- PCR revealed that the genome of Arresten - transferred cells contained a 449bp specific fragment of Arresten gene. Successful protein expression in supernatants was confirmed by Western blotting. CCK - 8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group （P 〈 0. 01 ）. Transwell＇s chamber showed that the number of control group, pSecTag,2 transfected group and pSecTag2 - AT transfected group were 28.70 ＋ 3.97, 26. 10 ± 4. 53 and 14.00 ±3.33 （P〈0.01）. CONCLUSION： Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.

关 键 词：真核表达 ARRESTEN 血管平滑肌细胞 细胞增殖 细胞运动 

分 类 号：R363[医药卫生—病理学]