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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 pathogenesis of placental lesions in pregnant women with preeclampsia, we simulated ischemia-reperfusion microenvironment in vivo and established a model of hypoxia-reoxygenation of HTR8 / SVneo in vitro to explore the induction of autophagy by hypoxia-reoxygenation And the impact on cell growth. The experiment was divided into control group, hypoxia-reoxygenation group and autophagy inhibitor 3-MA + hypoxia-reoxygenation group. Acridine orange staining and LC3-Ⅱ immunofluorescence staining were used to detect the level of autophagy, MTT The proliferation of Beclin-1 and LC3-Ⅱ was detected by Real time PCR and the expression of autophagy protein by Western blot. The results showed that the autophagy level of HTR8 / SVneo cells was significantly increased in hypoxia-reoxygenation group (p <0.01), and the expression of LC3-Ⅱ and Beclin-1 protein was significantly increased (p <0.01) (P <0.01). However, the autophagy of cells in hypoxia-reoxygenation group was significantly inhibited (P <0.01), the expression of Beclin-1 and LC3-Ⅱ mRNA and protein were significantly decreased Ability improved significantly (p <0.01). This indicates that the trophoblast HTR8 / SVneo initiates autophagy in hypoxia-reoxygenation environment, which may affect the cell proliferation by inducing type II programmed death when autophagy is over-repressed. When autophagy is inhibited, the cell proliferation ability is obviously restored.