摘要
目的探究Zeste基因增强子同源物2(EZH2)对体外培养的神经干细胞增殖的调控作用及潜在机制。方法采取第3~6代正常人神经干细胞,分别利用慢病毒构建稳转细胞株:EZH2基因过表达细胞(OV-EZH2)及对照细胞(OV-Control)、EZH2基因shRNA敲降细胞(sh-EZH2)及对照细胞(sh-Control)。CCK-8法和EdU染色法检测4种细胞的活力,流式细胞术检测细胞周期及凋亡,Western blot和实时荧光定量聚合酶链反应(qPCR)检测相关蛋白表达水平。结果与OV-Control细胞相比,OV-EZH2细胞48、72、96h时细胞增殖水平上调,且差异有统计学意义(P<0.05);与sh-Control细胞相比,sh-EZH2细胞在48、72、96h时细胞增殖水平下调,且差异有统计学意义(P<0.05);EZH2基因敲降造成细胞周期阻滞在G1期(P<0.05),而OV-EZH2加速细胞进入细胞周期S和G2期(P<0.05);EZH2干扰或过表达对细胞凋亡无明显影响(P>0.05);同时,与OV-Control细胞相比,OV-EZH2细胞增殖相关蛋白CyclinD1和c-Myc蛋白表达水平上调,且差异有统计学意义(P<0.05)。结论 EZH2基因能够通过促进CyclinD1和c-Myc蛋白表达水平上调,促进神经干细胞的增殖。
Objective To study the proliferative effect and mechanism of EZH2 on neural stem cells(NSCs)in vitro.Methods The third to sixth passage normal NSCs were adherent cultured.Lentivirus was used to construct stable cell lines:EZH2 overexpression cells(OV-EZH2),control cells(OV-Control),EZH2 shRNA knockdown cells(sh-EZH2)and control cells(sh-Control).Cell viability was detected by CCK-8 and EdU assay.Cell cycle and apoptosis were measured by flow cytometry.Western blot and qPCR were used to detect the expression of related proteins.Results Compared with the OV-Control cells,OV-EZH2 significantly promotes the proliferation of NSCs(P<0.05).Meanwhile,compared with the sh-Control cells,sh-EZH2 significantly decreased the proliferation of NSCs(P<0.05).sh-EZH2 resulted in G1/S phase delay(P<0.05),and OV-EZH2 accelerated cells to S and G2 phase transition(P<0.05).Nevertheless,EZH2 knockdown and overexpression exhibited no effect on the apoptosis of NSCs.Compared with OV-Control cells,OVEZH2 significantly increased the expression of Cyclin D1 and c-Myc(P<0.05).Conclusion EZH2 may promote proliferation of NSCs through Cyclin D1 and c-Myc pathway.
引文
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