外源性硫化氢对肝癌细胞系HepG2自噬的影响

doi:10.3969/j.issn.1006-7795.2015.01.022

首都医科大学学报 ›› 2015, Vol. 36 ›› Issue (1): 116-120.doi: 10.3969/j.issn.1006-7795.2015.01.022

外源性硫化氢对肝癌细胞系HepG2自噬的影响

陈宁¹, 王珊珊², 陈玉涵¹, 殷继明², 陈德喜², 丁惠国¹

1. 首都医科大学附属北京佑安医院肝病消化中心, 北京 100069;
2. 北京市肝病研究所, 北京 100069

收稿日期:2014-11-21 出版日期:2015-02-21 发布日期:2015-01-31
通讯作者: 丁惠国 E-mail:dinghuiguo@medmail.com.cn
基金资助:
国家自然科学基金(30872225);北京市科技计划(Z111107058811067);北京市卫生局高层次卫生人才学科带头人(2011-2-19).

Effects of exogenous hydrogen sulfide on autophagy of HepG2 cells

Chen Ning¹, Wang Shanshan², Chen Yuhan¹, Yin Jiming², Chen Dexi², Ding Huiguo¹

1. Department of Gastroenterology and Hepatology, Beijing You'an Hospital, Capital Medical University, Beijing 100069, China;
2. Beijing Institute of Hepatology, Beijing 100069, China

Received:2014-11-21 Online:2015-02-21 Published:2015-01-31
Supported by:
This study was supported by National Natural Science Foundation of China (3087225); Beijing Municipal Science and Technology Project(Z111107058811067); Beijing High-Level Talent Academic Leader/Personnel Training Programs awarded to Ding HG (2011-2-19).

摘要/Abstract

摘要： 目的探讨外源性硫化氢(hydrogen sulfide, H₂S)对肝癌细胞系HepG2细胞自噬的影响及其机制.方法应用外源性H₂S供体——硫氢化钠(sodium hydrosulfide, NaHS)处理HepG2细胞,采用Western blotting方法检测自噬相关蛋白微管相关蛋白轻链3-Ⅱ (microtubule-associated light chain 3-Ⅱ, LC3-Ⅱ)表达变化,Real-time PCR方法检测自噬相关基因beclin1和atg5的mRNA表达浓度,免疫荧光方法观察HepG2细胞自噬颗粒变化、凋亡相关蛋白M30及凋亡小体变化,AnnexinV/PI双标记流式细胞术检测细胞凋亡情况.结果瞬时转染绿色荧光蛋白(green fluorescent protein, GFP)-LC3质粒后,发现NaHS处理组HepG2细胞内GFP-LC3Ⅱ绿色荧光斑点明显增加,M30阳性细胞率[(8.37±1.03)%],与对照组[(2.14±0.69)%]比较差异有统计学意义(P<0.05);同时,高倍镜下观察结果显示,NaHS处理后细胞核异染色质明显增加,凋亡小体形成.AnnexinV-FITC/PI双染结果显示NaHS处理组细胞凋亡率高于对照组[(16.32±0.28)%vs(0.67±0.09)%], 差异有统计学意义(P<0.05).Real-time PCR显示NaHS可增加HepG2细胞beclin1和atg5 mRNA表达水平,LC3-Ⅱ表达明显增加.且自噬特异性抑制剂3-甲基腺嘌呤 (3-methyladenine, 3-MA)可降低NaHS所致的beclin1和atg5的mRNA表达.结论外源性硫化氢能够上调自噬相关基因beclin1和atg5表达,进而促进HepG2细胞发生自噬,并诱导细胞凋亡.

关键词: 硫化氢, 自噬, 自噬蛋白微管相关蛋白轻链3-Ⅱ, 凋亡

Abstract: Objective To investigate the effects of exogenous hydrogen sulfide (H₂S) on the autophagy of HepG2 cells and its underlying mechanism.Methods HepG2 cells were administrated by sodium hydrosulfide (NaHS), a donor of H₂S, for 24h, the expression of autophagy-related protein LC3-Ⅱ was detected via Western blotting; The mRNA level of autophagy related gene beclin1 and atg5 were detected via RT-PCR. The autophagy particles and apoptosis were observed using immunofluorescence. AnnexinV/PI flow cytometry were performed to assess the effect of NaHS on cell apoptosis. Results NaHS enhanced the autophagy-related protein (LC3-Ⅱ) expression and beclin1 mRNA, atg5 mRNA and autophagosome formation compared with control. 3-MA could inhibit NaHS-induced autophagy.Immunofluorescence showed the M30 positive cells were significantly increased compared with the controls.Additionally, both the nuclear heterochromatin and apoptotic bodies were significantly increased. Flowcytometry showed that NaHS increased the apoptosis rate of HepG2 cells. Conclusion The autophagy is induced by exogenous hydrogen sulfide through the autophagy related genes (beclin1 and atg5), and can promote apoptosis in HepG2 cells.

Key words: hydrogen sulfide, autophagy, microtubule-associated light chain 3-Ⅱ(LC3-Ⅱ), apoptosis

中图分类号:

R969.9

陈宁, 王珊珊, 陈玉涵, 殷继明, 陈德喜, 丁惠国. 外源性硫化氢对肝癌细胞系HepG2自噬的影响[J]. 首都医科大学学报, 2015, 36(1): 116-120.

Chen Ning, Wang Shanshan, Chen Yuhan, Yin Jiming, Chen Dexi, Ding Huiguo. Effects of exogenous hydrogen sulfide on autophagy of HepG2 cells[J]. Journal of Capital Medical University, 2015, 36(1): 116-120.

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外源性硫化氢对肝癌细胞系HepG2自噬的影响

Effects of exogenous hydrogen sulfide on autophagy of HepG2 cells

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