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

摘要

Objective To establish a miPSC-EC (Mouse induced pluripotent stem cell-Endothelial cell) line with stable YAP gene overexpression and lay a foundation for the studies of miPSC-EC with YAP overexpression in vitro and in vivo. Methods Reconstruct the plasmid pCMV-Flag-YAP2-5SA to recombinant lentiviral expression plasmid pPGK-2Flag-YAP2-Puro.Package the pPGK-2Flag-YAP2-Puro plasmid and packaging plasmids into mature lentivirus to infect 293FT cells. The supernatant of the infected cells was harvested to infect endothelial cells derived from miPSC and a stably infected cell line miPSC-EC/YAP were screened by puromycin. The YAP expression of miPSC-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 miPSC had the same morphology of endothelial cells. Almost all cells expressed endothelial cell marker CD31.The expression level of YAP mRNA in miPSC-EC/YAP was obviously higher than that of the nontransfected cells. The YAP protein in miPSC-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 miPSC is successfully induced. The cell line miPSC-EC/YAP with stable YAP gene overexpression is established successfully.%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（miPSC－EC）系，为 miPSC－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（pCMV－Flag－YAP2－5SA）改建成慢病毒表达质粒（pPGK－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miPSC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miPSC－EC，嘌呤霉素筛选稳定感染的细胞系 miPSC－EC／YAP，并用 RT－PCR 及 Western blot 方法验证。结果慢病毒表达质粒（pPGK－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。miPSC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miPSC－EC 中YAP mRNA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miPSC分化为EC，成功建立YAP基因稳定感染细胞系miPSC－EC／YAP。