腺苷通过内质网应激途径诱导HepG2细胞凋亡的研究  

The research of adenosine induced HepG2 cell apoptosis through endoplasmic reticulum stress pathway

作　　者：叶艳清[1] 李国平 蒲泽锦[1] 黄官友[2] 冯家琳[3] 韦碧柳[1] 吴灵飞[1] 

机构地区：[1]汕头大学医学院第二附属医院消化内科,广东汕头515041 [2]汕头大学医学院第二附属医院妇产科,广东汕头515041 [3]汕头大学医学院第二附属医院信息科,广东汕头515041

出　　处：《中国药理学通报》2010年第5期596-601,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30972925)

摘　　要：目的探讨腺苷(ADO)诱导人类肝癌HepG2细胞凋亡的分子机制。方法将不同浓度的ADO(0～6mmol.L-1)作用于HepG2细胞36h,采用MTT法测定ADO抑制细胞增殖效应。将HepG2细胞暴露于不同浓度的ADO(0～4mmol.L-1)作用36h或2mmol.L-1ADO作用不同时间(0～48h),观察细胞核的形态学改变;观察2mmol.L-1ADO处理12h和24h后细胞周期的变化;观察2mmol.L-1ADO作用前后Caspase-3和CHOP的亚细胞定位的变化;用West-ernblot检测不同浓度ADO作用后HepG2细胞Caspase-3,Caspase-4,CHOP,JNK的蛋白表达变化。结果ADO对HepG2细胞生长有明显的抑制作用,不同浓度ADO(0.5,1,2,4,6mmol.L-1)处理HepG2细胞36h后,与对照组相比,相对细胞存活数分别下降13.48%±0.12%,27.92%±0.25%,35.21%±0.42%,51.46%±0.24%,71.42%±0.58%,呈现剂量依赖性;不同浓度ADO作用36h或2mmol.L-1ADO作用不同时间(0～48h)后,随着ADO浓度的增加或作用时间的延长,HepG2细胞核发生典型核固缩、核碎裂、核分解等凋亡形态学改变;2mmol.L-1ADO作用12h或24h后,细胞周期分析出现亚二倍体峰,提示细胞发生凋亡,对照组、ADD处理12h及24h组细胞凋亡率分别为1.55%±0.12%、10.96%±0.07%和21.04%±0.26%;2mmol.L-1ADO诱导Caspase-3和CHOP表达增加,并从胞质易位进入胞核内;随着ADO浓度的升高,Caspase-4,Caspase-3,CHOP的表达均升高,均呈现剂量依赖性;而JNK的表达则没有变化。结论腺苷诱导HepG2细胞凋亡与内质网应激途径有关。Aim To investigate the role of endoplasmic reticulum stress in adenosine induced HepG2 cell apoptosis.Methods HepG2 cells were treated with different concentrations of ADO for 36 h,and the effect of ADO on cell proliferation was measured by MTT assay.Cell nuclei DAPI staining was used to detect the nuclei change after being treated with different concentrations ADO for 36 h or 2.0 mmol·L^-1 ADO for different time.The effect of ADO on HepG2 cell cycle was analysed by flow cytometry after being treated with 2.0 mmol·L^-1 ADO for 12 h or 24 h.Translocation of CHOP and Caspase-3 were measured by immunofluorescence after being treated with 2.0 mmol·L^-1 ADO for 36 h.The proteins expressions of CHOP,Caspase-4,Caspase-3 and JNK were assayed by western blot.Results The viability of HepG2 cell decreased in a dose-dependent manner;the relative cell viability of 0.5,1,2,4,6 mmol·L^-1 decreased by 13.48%±0.12%,27.92%±0.25%,35.21%±0.42%,51.46%±0.24%,71.42%±0.58%,compared with the control group respectively.The nuclei of HepG2 cells treated with different ADO concentrations or 2.0 mmol·L^-1 ADO showed condensation,rounding and shrinkage,then fragmentation,which demonstrated cell apoptosis.After being treated with 2.0 mmol·L^-1 ADO for 12 h or 24 h,cell cycle analysis showed sub-G1 phase increased;the apoptotic ratio of control,12 h and 24 h was 1.55%±0.12%,10.96%±0.07% and 21.04%±0.26% respectively.Immunofluorescence assay showed the CHOP and Caspase-3 translocation to the nuclei after being treated with 2.0 mmol·L^-1 ADO.The expressions of CHOP,Caspase-3 and Caspase-4 increased after being treated with different concentrations ADO for 36 h,while the expression of JNK did not change.Conclusion Endoplasmic reticulum stress is involved in adenosine-induced HepG2 cell apoptosis.

关 键 词：腺苷 细胞凋亡 内质网应激 HEPG2细胞 Caspase-3、4 CHOP 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R329.25[医药卫生—基础医学]