缺氧复氧诱导自噬基因表达对滋养细胞生长的影响与机制

Effect and Mechanism of Expression Autophagic Gene Induced by Hypoxia/Reoxygenation on Trophoblast Cell Proliferation

为探索子痫前期孕妇胎盘病变的发病机制,我们模拟体内缺血再灌注微环境,在体外建立胎盘滋养细胞HTR8/SVneo缺氧复氧模型,以探究缺氧复氧对细胞自噬的诱导作用及对细胞生长的影响。将实验分为对照组、缺氧复氧组及自噬抑制剂3-MA+缺氧复氧组,应用吖啶橙染色及LC3-Ⅱ免疫荧光染色检测经处理24 h后细胞自噬水平,MTT法检测细胞增殖能力,Real time PCR检测自噬基因Beclin-1、LC3-Ⅱ的表达,Western blot分析相应自噬蛋白的表达。结果显示缺氧复氧组HTR8/SVneo细胞自噬水平明显升高(p<0.01),伴随Beclin-1、LC3-Ⅱ基因及蛋白表达显著增高(p<0.01),细胞增殖同时显著受抑(p<0.01),而加入3-MA后缺氧复氧组细胞自噬水平明显受抑(p<0.01),Beclin-1、LC3-Ⅱ基因及蛋白表达显著下降(p<0.01),细胞增殖能力显著提高(p<0.01)。这表明滋养细胞HTR8/SVneo在缺氧复氧环境中启动细胞自噬,过度自噬时可能通过诱导Ⅱ型程序性死亡影响细胞增殖,当自噬被抑制后,细胞增殖能力明显恢复。

To explore the pat hogenesis of placental lesions of preeclampsia,we simulated ischemia and reperfusion in vivo microenvironment,established hypoxia/reoxygenation model of placental trophoblast HTR8/SVneo cells in vitro,in order to explore effect of autophagy and proliferation on cell induced by hypoxia/reoxygenation.The experiment was divided into control group,hypoxia/reoxygenation group and autophagy inhibitor 3-MA +hypoxia/reoxygenation group. Autophagy was detected by acridine orange staining and LC3-Ⅱ immunofluorescence staining after treating 24 h. Cell proliferation was analyzed by MTT assay. Expression of Beclin-1 and LC3-Ⅱwas detected by real time PCR and western blot. The results showed that cell autophagy were significantly increased in hypoxia/reoxygenation group(p<0.01),with upregulated of Beclin-1 and LC3-Ⅱ(p<0.01),proliferation was significantly inhibited. However autophagy significantly inhibited in 3-MA+ hypoxia/reoxygenation group,with downregulated of Beclin-1 and LC3-Ⅱ(p<0.01),proliferation was significantly improve(p<0.01). This indicates that autophagy has been induced in trophoblastic HTR8/SVneo cells by hypoxia/reoxygenation environment;excessive autophagy could weaken cell proliferation by inducing type Ⅱ cell death. When autophagy is inhibited,cell proliferation will restore.