miR-21調節缺血缺氧誘導的大鼠肝卵圓細胞自噬的研究

高勝強，陳冬冬，黃立棟，戴瑞杰，胡偉建，余華軍，張啟瑜，單云峰(溫州醫科大學附屬第一醫院肝膽外科，浙江溫州325000)

?

miR-21調節缺血缺氧誘導的大鼠肝卵圓細胞自噬的研究

高勝強，陳冬冬，黃立棟，戴瑞杰，胡偉建，余華軍，張啟瑜，單云峰
(溫州醫科大學附屬第一醫院肝膽外科，浙江溫州325000)

[摘 要]目的 探討miR-21在缺血缺氧誘導的大鼠肝卵圓細胞自噬中的作用。方法 體外培養肝卵圓細胞，慢病毒轉染肝卵圓細胞構建穩定的細胞株，分別提取各組細胞RNA逆轉錄后行SYBR Green實時熒光定量PCR，檢測各組miR-21的表達，以轉染好的穩定的細胞建立缺血缺氧模型，共分為3組:miR-21空載體慢病毒轉染HOC自噬組，miR-21增強慢病毒載體轉染HOC自噬組，miR-21-inhibition慢病毒載體轉染HOC自噬組。Hoechst 33258染色觀察細胞凋亡；應用丹(磺)酰戊二胺(Monodansylcadaverine，MDC)染色熒光定位法、觀察各組細胞的自噬；免疫印跡法(Western blotting)檢測各組細胞LC3-II/I蛋白表達情況。結果與空載體組比，增強組miR-21基因水平表達增強，而MDC染色減弱(自噬減少)，蛋白LC3-II/LC3-I的比值減少；而抑制組miR-21基因水平表達減少，MDC染色增強(自噬增強)，蛋白LC3-II/LC3-I的比值增多。結論 抑制miR-21過表達可以增強缺血缺氧引起的肝卵圓細胞的自噬，有利于肝卵圓細胞在缺血缺氧微環境中穩定細胞內環境，維持細胞的存活。

[關鍵詞]microRNA-21；自噬；肝卵圓細胞

Research of miR-21 on ischemia and hypoxia-induced autophagy modulation in rat hepatic oval cell GAO Sheng-qiang, CHEN Dong-dong, HUANG Li-dong, DAI Rui-jie, HU Wei-jian, YU Hua-jun, ZHANG Qi-yu, SHAN Yun-feng.Department of Hepatobiliary Surgery, the First Af■liated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China

Abstract ObjectiveTo investigate the role of miR-21 in ischemia-thypoxiainduced autophagic model of rat hepatic oval cells.MethodsHepatic 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 determine cell apoptosis.MDC (Monodansylcadaverine)-labeled autophagic vacuoles were observed under ■uorescent 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.ResultsCompared with the empty vector group, the expression of miR-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-II/LC3-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 microenvironment and maintain survival and the stability of cells.

Key words microRNA-21； autophagy； hepatic oval cell

肝卵圓細胞被認為是原始的兼性多能干細胞，目前普遍認為卵圓細胞位于小葉間膽管或Hering管，即終末膽管，也是門靜脈周圍肝細胞和膽管細胞的過渡區域。自噬(autophagy)是將細胞內受損、變性或者衰老的蛋白質以及受損細胞運輸到溶酶體進行消化降解的過程，是細胞應對營養缺乏、細胞密度負荷、低氧、氧化應激、感染等惡劣環境的生存方式之一?！?br>