人arresten基因的真核表达及其对血管平滑肌细胞增殖的影响  

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

作　　者：尚丹[1] 郑启昌[1] 宋自芳[1] 舒晓刚[1] 汪谢丹[1] 郭兴军[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院普外科,武汉430022

出　　处：《中华实验外科杂志》2008年第2期155-157,273,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30371396、30271242）

摘　　要：目的真核表达人arresten蛋白，并观察其对血管平滑肌细胞体外增殖的影响。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；收集浓缩转染48h细胞上清液，Western blot检测目的蛋白的表达。离体培养大鼠血管平滑肌细胞，用CCK-8法检测转染细胞上清液对平滑肌细胞体外增殖的影响。结果重组质粒pSecTag2-AT转染的COS-7细胞有目的基因mRNA的表达；转染细胞上清液中有arresten蛋白的表达。细胞体外增殖分析显示转染细胞上清液可显著抑制血管平滑肌细胞的体外增殖（F=40．154，P〈0．01）。结论真核表达的人arresten蛋白能有效抑制血管平滑肌细胞的体外增殖，为日后开展抑制血管新生内膜增生的基因治疗奠定了基础。Objective To express human arresten protein in eukaryotic cells, and investigate its effect on the proliferation in vitro of cultured vascular smooth cells. Methods COS-7 cells were transfeeted with recombinant eukaryotic expression plasmid pSeeTag2-AT mediated by liposome. And 48 h after transfeetion, reverse transeription-polymerase chain reaction （RT-PCR） and Western blot were used to detect the expression of arresten mRNA and protein in cells in concentrated supematants, respectively. Primary VSMCs of male Sprague-Dawley rats were cultured using the tissue explant method; Proliferation of VSMCs co-cultured with the concentrated supematants was detected using cell counting kit-8 （CCK-8） in vitro. Results RT-PCR revealed that the genome of arresten-transferred cells contained a 449 bp specific fragment of arresten gene. Successful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by arresten protein as compared with control cells （ F = 40. 154, P 〈 0.01 ）. Conclusion Arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide foundation for the research of gene therapy.

关 键 词：血管平滑肌细胞 细胞增殖 基因表达 

分 类 号：R543[医药卫生—心血管疾病]