黑色素瘤抗原基因3在K562细胞内质网应激反应性凋亡中的表达及意义  

Expression and Significance of Melanoma Antigen Gene-3 in Endoplasmic Reticulum Stress-Induced Apoptosis of K562 Cells

作　　者：冯献启[1] 肖娟[1] 聂淑敏[2] 刘芳[1] 游泳[1] 邹萍[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院血液学研究所,武汉430022 [2]滨州医学院附属医院功能检查科,滨州256603

出　　处：《中国实验血液学杂志》2005年第5期741-745,共5页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目;编号30370595

摘　　要：本研究旨在探讨黑色素瘤抗原基因3(melanomaantigengene3,MAGE3)在内质网应激反应性凋亡中的表达及意义。应用Ca2+荧光指示剂Fura2/AM通过荧光分光光度计测定白血病细胞系K562及其多药耐药细胞株K562/A02经thapsigargin作用前后细胞内Ca2+浓度([Ca2+]i)变化;Westernblot检测GRP78蛋白表达变化;荧光显微镜观察细胞凋亡形态变化;TUNEL法检测细胞凋亡率;RTPCR检测MAGE3基因mRNA水平表达变化。结果显示:①毒胡萝卜素(thapsigargin)诱导K562和K562/A02细胞[Ca2+]i出现不同程度的升高并呈一定的浓度依赖性,同时诱导GRP78蛋白表达增加以及K562和K562/A02细胞典型的凋亡改变,细胞凋亡率亦呈一定的浓度依赖性;在静息状态下K562/A02细胞[Ca2+]i较K562细胞明显增高;②毒胡萝卜素诱导K562和K562/A02细胞凋亡过程中,MAGE3mRNA表达明显下调;此外,与K562细胞比较,K562/A02细胞MAGE3mRNA表达明显上调。结论:①毒胡萝卜素在一定浓度范围内可诱导K562及其多药耐药细胞株K562/A02发生内质网应激反应性凋亡,并可能与MAGE3表达下调有关或MAGE3基因可能参与此凋亡途径的调控;②MAGE3可能具有抗凋亡活性,K562/A02细胞多药耐药性可能与细胞[Ca2+]i增高和MAGE3表达上调有关。This study was aimed to explore the expression and significance of melanoma antigen gene-3 （ MAGE-3 ） in endoplasmic reticulum stress-induced apoptosis, After the treatment of leukemia cell line K562 and its multidrugresistant cell line K562/A02 by thapsigargin, intracellular calcium concentrations （[ Ca^2＋ ] i） in K562 and K562/A02 were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM; expression changes of glucose-regulated protein 78 （ GRP78 ） were detected by Western blot; morphological change of apoptotic cell was investigated by AO/EB fluorescent staining under fluorescent microscope; apoptosis rate was determined by terminal deoxyribonucleotidyl transferase （TdT）-mediated dUTP nick end labelling （TUNEL） staining ; the expression of MAGE-3 gene mRNA was detected by RT-PCR. The results showed that （ 1 ） thapsigargin induced the enhancement of [ Ca^2＋ ] i with different extent in K562 and K562/A02 cells, and the enhancement of [ Ca^2＋] i was dose-dependent in experiment range, At the same time, thapsigargin induced upregulation of GRP78 protein expression and typical apoptotic changes of K562 and K562/A02 cells, apoptotic rate was also dose-dependent in experiment range. The [ Ca^2＋ ] i in K562/A02 cells were higher than that in K562 cells, （2） in the course of endoplasmic reficulum stressinduced apoptosis by thapsigargin, the expression of MAGE-3 gene mRNA was remarkably downregulated, Moreover, the expression of MAGE-3 gene mRNA in K562/A02 cells was higher than that in K562 cells, It is concluded that （ 1 ） thapsigargin induces endoplasmic reticulum stress-induced apoptosis of K562 and K562/A02 cells in experiment range, and this may be associated with downregulation of MAGE-3 mRNA expression or MAGE-3 gene may participates in the regulation of endoplasmic reticulum stress-induced apoptosis. （2） MAGE-3 gene may possess anti-apoptotic activity,multidrug resistance in K562/A02 cells can be associated with Ⅰ Ca^2�

关 键 词：MAGE-3基因 毒胡萝卜素 K562细胞 细胞内质网 细胞凋亡 

分 类 号：R733.7[医药卫生—肿瘤] R329.26[医药卫生—临床医学]