摘要
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 ischemia-thypoxiainduced autophagic model of rat hepatic oval cells. Methods Hepatic oval cells were cultured in vitro and a stable hepatic oval cells line was constructed by lentivirus （miR-21 enhanced lentivirus; miR-21 inhibited lentivirus; vector lentivirus）. The RNA was extracted from each group and reverstranscripted, then SYBR real-time PCR was conducted to detect the miR-21 expression. Simulated ischemia and hypoxia of transfected HOCs （hepatic oval cells） as an in vitro model of ischemic-hypoxic microenvironment were employed in this study. Hoechst 33258 staining was used to deter- mine cell apoptosis. MDC （Monodansylcadaverine）-labeled autophagic vacuoles were observed under fluorescent inverted phase contrast microscopy in HOC. The expression of LC3 and the result of LC3-I converted to LC3-II was detected with Western blotting. Results Compared with the empty vector group, the expression ofmiR-21 was increased in enhanced group and decreased in inhibited group; Compared with the empty vector group, enhanced group MDC staining was decreased, and protein LC3-IIFLC3-I was reduced. Compared with the empty vector, inhibited group MDC staining was enhanced, and the ratio of protein LC3-II/LC3-I was increased. Conclusion Inhibition of miR-21 expression can enhance autophagy in ischemia-hypoxiainduced model. Autophagy of HOC can be induced by the ischemic and hypoxic mieroenvironment and maintain survival and the stability of cells.
出处
《肝胆胰外科杂志》
CAS
2016年第1期46-50,共5页
Journal of Hepatopancreatobiliary Surgery
基金
浙江省自然科学基金项目(LY12H03006)
温州市科委课题(Y20110078)
