慢病毒介导Yes相关蛋白基因过表达促小鼠诱导性多能干细胞来源的内皮细胞增殖

Study of the proliferation of endothelial cells derived from mouse induced pluripotent stem cells by lentivirus - mediated Yes - associated protein gene overexpression

目的探讨慢病毒介导Yes相关蛋白（YAP）过表达促小鼠诱导性多能干细胞来源的内皮细胞（miPSC-EC）增殖的机制。方法诱导小鼠诱导性多能干细胞（miPSC）为内皮细胞（EC），慢病毒介导质粒pPGK-2Flag-YAP转染miPSC-EC，嘌呤霉素筛选出稳定转染细胞株miPSC-EC/YAP。Western blot验证YAP是否成功转染miPSC-EC，并检测转染YAP后miPSC-EC内人第10号染色体上缺失与张力蛋白同源的磷酸酯酶基因（PTEN）、磷酸化蛋白激酶B（p-Akt）等蛋白的表达，同时应用噻唑蓝（MTT）比色法检测YAP对miPSC-EC增殖的影响。结果miPSC诱导分化所得细胞形态符合EC特征，几乎都表达EC标志血小板内皮细胞黏附分子（CD31）。Western blot结果显示，转染YAP的miPSC-EC中YAP高表达（未转染细胞YAP几乎不表达），miPSC-EC/YAP组较未转染组PTEN相对表达量明显降低（0.635±0.017比0.879±0.034, P=0.023），p-Akt/相对表达量显著升高（0.450±0.025比0.073±0.018, P=0.007）。同步化之后24、48、72 h，miPSC-EC/YAP组吸光度（A）值较未转染组显著增加（0.113±0.004比0.077±0.004，P=0.000；0.368±0.010比0.199±0.006，P=0.000；0.716±0.004比0.418±0.018，P=0.000）。结论成功诱导miPSC为EC，建立过表达YAP的细胞株miPSC-EC/YAP。过表达YAP抑制PTEN的表达，提高Akt磷酸化。YAP可能通过参与磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/Akt）信号通路促进miPSC-EC的增殖。

ObjectivePreliminarily study the mechanism of the proliferation of mouse Induced pluripotent stem cells （miPSC）-endothelial cells （EC） by lentivirus-mediated yes-associated protein （YAP） gene overexpression.MethodsInduce miPSC to EC. The pPGK-2 Flag YAP plasmid was packaged into mature lentivirus to infect 293FT cells. The supernatant of the infected cells was harvested to infect miPSC-EC and a stably infected cell lines were screened by puromycin, in which YAP expression was detected using Western blotting. Detect and compare the protein expression level of Phosphatase and tensin homolog deleted on chromosome ten （PTEN） and increased the phosphorylation of protein kinase B （Akt）, phosphorylated-protein kinase B （p-Akt） between miPSC-EC and miPSC-EC/YAP. Cell proliferation ability was determined by methyl thiazolyl tetrazolium assay （MTT）.ResultsThe cells induced from miPSC have the same morphology of endothelial cells. Almost all cells expressed endothelial cell marker CD31 （Platelet endothelial cell adhesion molecule-1）. The YAP protein in miPSC-EC/YAP was obviously expressed, and while the YAP protein in nontransfected cells was almost not expressed. Overexpressing YAP in miPSC-EC resulted in the decreased expressing of PTEN （0.635±0.017 vs. 0.879±0.034, P=0.023） and the increased expressing of P-Akt （0.450±0.025 vs. 0.073±0.018, P=0.007）. The optical density （A） value of YAP group was higher than that in control group at time points of 24 h, 48 h and 72 h after synchronization （0.113±0.004 vs. 0.077±0.004, P=0.000; 0.368±0.010 vs. 0.199±0.006, P=0.000; 0.716±0.004 vs. 0.418±0.018, P=0.000）.ConclusionEC derived from miPSC was successfully induced. The cell line miPSC-EC/YAP with stable YAP gene overexpression was established successfully. Overexpressing YAP inhibited the expression of PTEN. It indicated that overexpressing YAP may promote the proliferation of miPSC-EC through the phoshoinositide-3-kinase/protein kinase B （PI3K-Akt） signal pathway.