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目的探讨miR-21在缺血缺氧诱导的大鼠肝卵圆细胞自噬中的作用。方法体外培养肝卵圆细胞,慢病毒转染肝卵圆细胞构建稳定的细胞株,分别提取各组细胞RNA逆转录后行SYBR Green实时荧光定量PCR,检测各组miR-21的表达,以转染好的稳定的细胞建立缺血缺氧模型,共分为3组:miR-21空载体慢病毒转染HOC自噬组,miR-21增强慢病毒载体转染HOC自噬组,miR-21-inhibition慢病毒载体转染HOC自噬组。Hoechst 33258染色观察细胞凋亡;应用丹(磺)酰戊二胺(Monodansylcadaverine,MDC)染色荧光定位法、观察各组细胞的自噬;免疫印迹法(Western blotting)检测各组细胞LC3-Ⅱ/Ⅰ蛋白表达情况。结果与空载体组比,增强组miR-21基因水平表达增强,而MDC染色减弱(自噬减少),蛋白LC3-Ⅱ/LC3-Ⅰ的比值减少;而抑制组miR-21基因水平表达减少,MDC染色增强(自噬增强),蛋白LC3-Ⅱ/LC3-Ⅰ的比值增多。结论抑制miR-21过表达可以增强缺血缺氧引起的肝卵圆细胞的自噬,有利于肝卵圆细胞在缺血缺氧微环境中稳定细胞内环境,维持细胞的存活。
Objective To investigate the role of miR-21 in autophagy induced by hypoxia-ischemia in rat hepatic oval cells. Methods Hepatic oval cells were cultured in vitro. The lentiviral vector was transfected into hepatic oval cells to construct a stable cell line. The RNA of each group was extracted and reverse transcribed. SYBR Green real-time PCR was used to detect the expression of miR-21. The cells stained with HIF-1αmRNA were subcloned into three groups: miR-21 empty vector lentivirus transfected HOC autophagy, miR-21 enhanced lentiviral vector transfected HOC autophagy, miR-21 -inhibition lentiviral vector transfected HOC autophagy. Hoechst 33258 staining was used to observe the cell apoptosis. Autophagy was observed by Monodansylcadaverine (MDC) staining. The expression of LC3-Ⅱ / Ⅰ protein expression. Results Compared with the empty vector group, the expression of miR-21 gene was enhanced in enhanced group, while the expression of miR-21 gene was decreased in MDC-treated group and decreased in LC-Ⅱ / LC3-Ⅰ group MDC staining enhanced (enhanced autophagy), protein LC3-Ⅱ / LC3-Ⅰ ratio increased. Conclusion Overexpression of miR-21 can enhance the autophagy of hepatic oval cells induced by hypoxia and hypoxia, and help the hepatic oval cells to stabilize the intracellular environment and maintain the survival of cells in the hypoxic-ischemic microenvironment.