首都医科大学学报
首都醫科大學學報
수도의과대학학보
JOURNAL OF CAPITAL UNIVERSITY OF MEDICAL SCIENCES
2015年
1期
116-120
,共5页
陈宁%王珊珊%陈玉涵%殷继明%陈德喜%丁惠国
陳寧%王珊珊%陳玉涵%慇繼明%陳德喜%丁惠國
진저%왕산산%진옥함%은계명%진덕희%정혜국
硫化氢%自噬%自噬蛋白微管相关蛋白轻链3-Ⅱ%凋亡
硫化氫%自噬%自噬蛋白微管相關蛋白輕鏈3-Ⅱ%凋亡
류화경%자서%자서단백미관상관단백경련3-Ⅱ%조망
hydrogen sulfide%autophagy%microtubule-associated light chain 3-Ⅱ(LC3-Ⅱ)%apoptosis
目的：探讨外源性硫化氢（hydrogen sulfide，H2 S）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H2 S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。
目的：探討外源性硫化氫（hydrogen sulfide，H2 S）對肝癌細胞繫HepG2細胞自噬的影響及其機製。方法應用外源性H2 S供體———硫氫化鈉（sodium hydrosulfide，NaHS）處理HepG2細胞，採用Western blotting方法檢測自噬相關蛋白微管相關蛋白輕鏈3-Ⅱ（microtubule-associated light chain 3-Ⅱ，LC3-Ⅱ）錶達變化，Real-time PCR方法檢測自噬相關基因beclin1和atg5的mRNA錶達濃度，免疫熒光方法觀察HepG2細胞自噬顆粒變化、凋亡相關蛋白M30及凋亡小體變化，AnnexinV／PI雙標記流式細胞術檢測細胞凋亡情況。結果瞬時轉染綠色熒光蛋白（green fluorescent protein，GFP）-LC3質粒後，髮現NaHS處理組HepG2細胞內GFP-LC3Ⅱ綠色熒光斑點明顯增加，M30暘性細胞率[（8.37±1.03）％]，與對照組[（2.14±0.69）％]比較差異有統計學意義（P＜0.05）；同時，高倍鏡下觀察結果顯示，NaHS處理後細胞覈異染色質明顯增加，凋亡小體形成。AnnexinV-FITC／PI雙染結果顯示NaHS處理組細胞凋亡率高于對照組[（16.32±0.28）％vs（0.67±0.09）％]，差異有統計學意義（P＜0.05）。Real-time PCR顯示NaHS可增加HepG2細胞beclin1和atg5 mRNA錶達水平，LC3-Ⅱ錶達明顯增加。且自噬特異性抑製劑3-甲基腺嘌呤（3-methyladenine，3-MA）可降低NaHS所緻的beclin1和atg5的mRNA錶達。結論外源性硫化氫能夠上調自噬相關基因beclin1和atg5錶達，進而促進HepG2細胞髮生自噬，併誘導細胞凋亡。
목적：탐토외원성류화경（hydrogen sulfide，H2 S）대간암세포계HepG2세포자서적영향급기궤제。방법응용외원성H2 S공체———류경화납（sodium hydrosulfide，NaHS）처리HepG2세포，채용Western blotting방법검측자서상관단백미관상관단백경련3-Ⅱ（microtubule-associated light chain 3-Ⅱ，LC3-Ⅱ）표체변화，Real-time PCR방법검측자서상관기인beclin1화atg5적mRNA표체농도，면역형광방법관찰HepG2세포자서과립변화、조망상관단백M30급조망소체변화，AnnexinV／PI쌍표기류식세포술검측세포조망정황。결과순시전염록색형광단백（green fluorescent protein，GFP）-LC3질립후，발현NaHS처리조HepG2세포내GFP-LC3Ⅱ록색형광반점명현증가，M30양성세포솔[（8.37±1.03）％]，여대조조[（2.14±0.69）％]비교차이유통계학의의（P＜0.05）；동시，고배경하관찰결과현시，NaHS처리후세포핵이염색질명현증가，조망소체형성。AnnexinV-FITC／PI쌍염결과현시NaHS처리조세포조망솔고우대조조[（16.32±0.28）％vs（0.67±0.09）％]，차이유통계학의의（P＜0.05）。Real-time PCR현시NaHS가증가HepG2세포beclin1화atg5 mRNA표체수평，LC3-Ⅱ표체명현증가。차자서특이성억제제3-갑기선표령（3-methyladenine，3-MA）가강저NaHS소치적beclin1화atg5적mRNA표체。결론외원성류화경능구상조자서상관기인beclin1화atg5표체，진이촉진HepG2세포발생자서，병유도세포조망。
Objective To investigate the effects of exogenous hydrogen sulfide (H2 S)on the autophagy of HepG2 cells and its underlying mechanism.Methods HepG2 cells were administrated by sodium hydrosulfide (NaHS),a donor of H2 S,for 24h,the expression of autophagy-related 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.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.
