摘要
目的探讨外源性硫化氢(hydrogen sulfide,H2S)对肝癌细胞系Hep G2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,Na HS)处理Hep G2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Hep G2细胞自噬颗粒变化、凋亡相关蛋白M30及凋亡小体变化,Annexin V/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Na HS处理组Hep G2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,Na HS处理后细胞核异染色质明显增加,凋亡小体形成。Annexin V-FITC/PI双染结果显示Na HS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Na HS可增加Hep G2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Na HS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Hep G2细胞发生自噬,并诱导细胞凋亡。
Objective To investigate the effects of exogenous hydrogen sulfide( H2S) on the autophagy of Hep G2 cells and its underlying mechanism. Methods Hep G2 cells were administrated by sodium hydrosulfide( Na HS),a donor of H2 S,for 24 h,the expression of autophagyrelated protein LC3-Ⅱwas 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. Annexin V/PI flow cytometry were performed to assess the effect of Na HS on cell apoptosis. Results Na HS enhanced the autophagy-related protein( LC3-Ⅱ) expression and beclin1 mRNA,atg5 mRNA and autophagosome formation compared with control. 3-MA could inhibit Na HS-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 Na HS increased the apoptosis rate of Hep G2 cells. Conclusion The autophagy is induced by exogenous hydrogen sulfide through the autophagy related genes( beclin1 and atg5),and can promote apoptosis in Hep G2 cells.
出处
《首都医科大学学报》
CAS
北大核心
2015年第1期116-120,共5页
Journal of Capital Medical University
基金
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)~~
关键词
硫化氢
自噬
自噬蛋白微管相关蛋白轻链3-Ⅱ
凋亡
hydrogen sulfide
autophagy
microtubule-associated light chain 3-Ⅱ(LC3-Ⅱ)
apoptosis