中华神经外科杂志
中華神經外科雜誌
중화신경외과잡지
Chinese Journal of Neurosurgery
2015年
4期
406-411
,共6页
目的 用自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)抑制自噬,观察其对顺铂诱导的U251细胞凋亡的影响及其作用机制.方法 MTT法检测3-MA对顺铂诱导的U251细胞生长抑制的影响;Hoechst33342染色,观察3-MA对顺铂诱导细胞凋亡的影响;蛋白免疫印迹法检测内质网应激(endoplasmic reticulum stress,ERS)相关蛋白,泛素化蛋白(ubquitinated proteins Ub1),蛋白二硫键异构酶(protein disulfide isomerase,PDI),葡萄糖调节蛋白78(glucose-regulated protein78,Grp78)的表达,并检测了C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(C/EBP homologous protein,CHOP),活化的Caspase-4和活化的Caspase-3的表达水平;免疫荧光染色后共聚焦显微镜观察3-MA与顺铂联合作用于U251细胞,微管相关蛋白轻链3(microtubule-associated protein 1 light chain 3,LC3)、PDI的表达.结果 MTT结果显示,3-MA可以促进顺铂对U251生长抑制(顺铂组与对照组相比,P=0.00324;联合组与顺铂组相比P=0.02249),Hoechst33342染色结果显示3-MA可以促进顺铂对U251凋亡诱导作用;免疫印迹法结果显示,3-MA增加了顺铂诱导的细胞内泛素化蛋白、ERS标志蛋白PDI、Grp78的表达(顺铂组与对照组相比,P值分别为0.00294,0.0012,0.00179;联合组与顺铂组相比,P值分别为0.01896,0.01515,0.00734);促进顺铂诱导的CHOP表达,增强了Caspase-4和Caspase-3的活化(顺铂组与对照组相比,P值分别为0.00081,0.00017,0.00018;联合组与顺铂组相比,P值分别为0.00671,0.00934,0.01124),3-MA可以有效抑制顺铂诱导的U251细胞LC3II的表达(顺铂组与对照组相比,P=0.00447;联合组与顺铂组相比,P=0.01588);免疫荧光染色结果显示,3-MA与顺铂联合作用于U251细胞,抑制了LC3的聚集、促进了PDI的表达.结论 3-MA通过抑制自噬,增强了顺铂诱导的ERS,从而促进顺铂诱导的U251细胞凋亡.
目的 用自噬抑製劑3-甲基腺嘌呤(3-methyladenine,3-MA)抑製自噬,觀察其對順鉑誘導的U251細胞凋亡的影響及其作用機製.方法 MTT法檢測3-MA對順鉑誘導的U251細胞生長抑製的影響;Hoechst33342染色,觀察3-MA對順鉑誘導細胞凋亡的影響;蛋白免疫印跡法檢測內質網應激(endoplasmic reticulum stress,ERS)相關蛋白,汎素化蛋白(ubquitinated proteins Ub1),蛋白二硫鍵異構酶(protein disulfide isomerase,PDI),葡萄糖調節蛋白78(glucose-regulated protein78,Grp78)的錶達,併檢測瞭C/EBP環燐痠腺苷反應元件結閤轉錄因子同源蛋白(C/EBP homologous protein,CHOP),活化的Caspase-4和活化的Caspase-3的錶達水平;免疫熒光染色後共聚焦顯微鏡觀察3-MA與順鉑聯閤作用于U251細胞,微管相關蛋白輕鏈3(microtubule-associated protein 1 light chain 3,LC3)、PDI的錶達.結果 MTT結果顯示,3-MA可以促進順鉑對U251生長抑製(順鉑組與對照組相比,P=0.00324;聯閤組與順鉑組相比P=0.02249),Hoechst33342染色結果顯示3-MA可以促進順鉑對U251凋亡誘導作用;免疫印跡法結果顯示,3-MA增加瞭順鉑誘導的細胞內汎素化蛋白、ERS標誌蛋白PDI、Grp78的錶達(順鉑組與對照組相比,P值分彆為0.00294,0.0012,0.00179;聯閤組與順鉑組相比,P值分彆為0.01896,0.01515,0.00734);促進順鉑誘導的CHOP錶達,增彊瞭Caspase-4和Caspase-3的活化(順鉑組與對照組相比,P值分彆為0.00081,0.00017,0.00018;聯閤組與順鉑組相比,P值分彆為0.00671,0.00934,0.01124),3-MA可以有效抑製順鉑誘導的U251細胞LC3II的錶達(順鉑組與對照組相比,P=0.00447;聯閤組與順鉑組相比,P=0.01588);免疫熒光染色結果顯示,3-MA與順鉑聯閤作用于U251細胞,抑製瞭LC3的聚集、促進瞭PDI的錶達.結論 3-MA通過抑製自噬,增彊瞭順鉑誘導的ERS,從而促進順鉑誘導的U251細胞凋亡.
목적 용자서억제제3-갑기선표령(3-methyladenine,3-MA)억제자서,관찰기대순박유도적U251세포조망적영향급기작용궤제.방법 MTT법검측3-MA대순박유도적U251세포생장억제적영향;Hoechst33342염색,관찰3-MA대순박유도세포조망적영향;단백면역인적법검측내질망응격(endoplasmic reticulum stress,ERS)상관단백,범소화단백(ubquitinated proteins Ub1),단백이류건이구매(protein disulfide isomerase,PDI),포도당조절단백78(glucose-regulated protein78,Grp78)적표체,병검측료C/EBP배린산선감반응원건결합전록인자동원단백(C/EBP homologous protein,CHOP),활화적Caspase-4화활화적Caspase-3적표체수평;면역형광염색후공취초현미경관찰3-MA여순박연합작용우U251세포,미관상관단백경련3(microtubule-associated protein 1 light chain 3,LC3)、PDI적표체.결과 MTT결과현시,3-MA가이촉진순박대U251생장억제(순박조여대조조상비,P=0.00324;연합조여순박조상비P=0.02249),Hoechst33342염색결과현시3-MA가이촉진순박대U251조망유도작용;면역인적법결과현시,3-MA증가료순박유도적세포내범소화단백、ERS표지단백PDI、Grp78적표체(순박조여대조조상비,P치분별위0.00294,0.0012,0.00179;연합조여순박조상비,P치분별위0.01896,0.01515,0.00734);촉진순박유도적CHOP표체,증강료Caspase-4화Caspase-3적활화(순박조여대조조상비,P치분별위0.00081,0.00017,0.00018;연합조여순박조상비,P치분별위0.00671,0.00934,0.01124),3-MA가이유효억제순박유도적U251세포LC3II적표체(순박조여대조조상비,P=0.00447;연합조여순박조상비,P=0.01588);면역형광염색결과현시,3-MA여순박연합작용우U251세포,억제료LC3적취집、촉진료PDI적표체.결론 3-MA통과억제자서,증강료순박유도적ERS,종이촉진순박유도적U251세포조망.
Objective 3-methyl adenine (3-methyladenine,3-MA) was used to test the effect on cisplatin induced U251 cell apoptosis.Methods MTT assay was used to test the effect of 3-MA on cisplatin cytotoxicity in U251 cells.Hoechst33342 staining was used to test the effect of 3-MA on cisplatin induced apoptosis.Western bolt was used to detect the expression of ER stress associated proteins,ubiquitinated protein,protein disulfide isomerase (PDI),glucose-regulated protein78 (Grp78),C/EBP homologous protein (CHOP),and activation of caspase-4 and caspase-3.The expression of microtubule-associated protein 1 light chain 3 (LC3) and PDI were observed by immunofluorescence staining and confocal microscopy.Results MTT assay showed that 3-MA could enhance cisplatin induced growth inhibition rate in human glioma U251 cells (P =0.00324 cisplatin troup vs.control group;P =0.02249 3-MA + cisplatin group vs.cisplatin group).Hoechst33342 staining showed that 3-MA could increase cisplatin induced apoptotic effect in U251 cells.Western blot showed that 3-MA increases the expressions of ubiquitinated proteins,ERS marker proteins PDI,Grp78 (P =0.00294,0.0012,0.00179 cisplatin group vs.control group;P =0.01896,0.01515,0.00734 3-MA + cisplatin group vs cisplatin group),CHOP,and active Caspase-4 and active Caspase-3 (P =0.00081,0.00017,0.00018 cisplatin group vs.control group;P =0.00671,0.00934,0.01124 3-MA + cisplatin group vs.cisplatin group).3-MA could inhibit the expression of LC3II induced by cisplatin in U251 cell (P =0.00447 cisplatin group vs.control group;P =0.01588 3-MA + cisplatin group vs.cisplatin group).Indirect immunofluorence staining showed that 3-MA inhibited the aggregation of LC3 and increased the expression of PDI.Conclusion 3-MA combined with cisplatin enhances cisplatin induced apoptosis by increasing ER stress.
