慢病毒介导的miPSC-EC YAP基因过表达及稳定感染细胞系的建立  

Establishment of a mi PSC-EC line with stable YAP gene overexpression by lentivirus-mediated YAP overexpressing

作　　者：张雪[1] 郝长宁[1] 曾庆坛 阚科佳 陈佳全[1] 吴圣俊 严泽振[1] 张岚[1] 

机构地区：[1]上海交通大学医学院附属仁济医院血管外科,上海200127

出　　处：《中国血管外科杂志（电子版）》2016年第1期28-33,共6页Chinese Journal of Vascular Surgery(Electronic Version)

基　　金：上海市科委国际科技合作基金项目(14430721400)

摘　　要：目的建立稳定过表达Yes相关蛋白(YAP)的小鼠诱导性多能干细胞来源的内皮细胞(miP SC-EC)系,为miP SC-EC过表达YAP基因的体内外实验研究奠定基础。方法将模板质粒(p CMV-Flag-YAP2-5SA)改建成慢病毒表达质粒(p PGK-2Flag-YAP2-Puro);将慢病毒表达质粒与包装质粒组成共包装系统转染293FT细胞;包装慢病毒,收集病毒原液,超滤浓缩,测定滴度。诱导miP SC分化为EC,观察其形态特征,并行组织化学鉴定。包装好的慢病毒感染miP SC-EC,嘌呤霉素筛选稳定感染的细胞系miP SC-EC/YAP,并用RT-PCR及Western blot方法验证。结果慢病毒表达质粒(p PGK-2Flag-YAP2-Puro)构建成功,并成功包装YAP基因过表达慢病毒,滴度达到1.2×109 TU/ml。miP SC诱导分化所得细胞符合EC形态特征,几乎都表达EC标志CD31。转染YAP的miP SC-EC中YAP m RNA表达水平较未转染细胞明显升高,YAP蛋白高表达(未转染细胞中YAP蛋白几乎不表达)。结论成功构建YAP基因慢病毒过表达载体,诱导miP SC分化为EC,成功建立YAP基因稳定感染细胞系miP SC-EC/YAP。Objective To establish a miP SC-EC（Mouse induced pluripotent stem cell-Endothelial cell） line with stable YAP gene overexpression and lay a foundation for the studies of miP SC-EC with YAP overexpression in vitro and in vivo. Methods Reconstruct the plasmid p CMV-Flag-YAP2-5SA to recombinant lentiviral expression plasmid p PGK-2Flag-YAP2-Puro.Package the p PGK-2Flag-YAP2-Puro plasmid and packaging plasmids into mature lentivirus to infect 293 FT cells. The supernatant of the infected cells was harvested to infect endothelial cells derived from miP SC and a stably infected cell line miP SC-EC / YAP were screened by puromycin. The YAP expression of miP SC-EC / YAP was detected by real-time PCR and Western blot. Results The lentivirus carrying YAP was constructed successfully and with a virus titer of 1.2×109TU / ml. The cells induced from miP SC had the same morphology of endothelial cells. Almost all cells expressed endothelial cell marker CD31.The expression level of YAP m RNA in miP SC-EC / YAP was obviously higher than that of the nontransfected cells. The YAP protein in miP SC-EC / YAP was obviously expressed,and while the YAP protein in nontransfected cells was almost not expressed.Conclusion Lentivirus vector of YAP gene overexpressing is successfully constructed. EC derived from miP SC is successfully induced. The cell line miP SC-EC / YAP with stable YAP gene overexpression is established successfully.

关 键 词：Yes相关蛋白 慢病毒 诱导性多能干细胞 内皮细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]