衣霉素诱导大鼠心肌细胞内质网应激凋亡模型的构建  

Construction of Neonatal Rat Cardiomyocyte Apoptosis Model by Tunicamycin-Induced Endoplasmic Reticulum Stress

作　　者：沈明志[1] 刘佳妮[1] 翟雅莉[1] 赵萌[1] 丁铭格[1] 王博[1] 岳劲[1] 王晓明[1] 

机构地区：[1]第四军医大学西京医院老年病科,陕西西安710032

出　　处：《现代生物医学进展》2011年第5期801-804,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目资助(30770847)

摘　　要：目的:通过衣霉素诱导内质网应激建立新生大鼠心肌细胞凋亡模型。方法:不同浓度、不同时间的衣霉素作用于原代培养乳鼠心肌细胞,通过MTT实验和流式细胞术测定心肌细胞的存活率和凋亡率,Western blot检测内质网应激蛋白GRP78,CHOP表达水平。结果:①与阴性对照组相比,衣霉素具有损伤心肌细胞的作用,并呈现剂量与时间依赖关系(P<0.05,n=12)。②通过流式细胞术判断心肌细胞死亡的性质,当衣霉素浓度为100ng/ml,作用72h时,心肌细胞存活率和凋亡率分别为57.4±3.2%(n=12),25.9±5.8%(n=3)。提示衣霉素损伤细胞的形式主要为凋亡性死亡。③内质网应激蛋白GRP78和CHOP表达于6h开始增加,24h达到峰值,随后呈下降趋势。结论:应用衣霉素成功诱导SD乳鼠心肌细胞内质网应激凋亡模型,衣霉素的最佳诱导浓度为100ng/ml,作用时间为72h。Objective：To establish endoplasmic reticulum stress-induced apoptotic model by tunicamycin induced on cultured neonatal rat cardiomyocytes.Methods：Neonatal rat cardiomyocytes in primary culture were exposed to tunicamycin with different concentrations and action times.MTT assay and flow cytometry analysis were applied to detect cardiomyocyte viability and apoptosis rate.Western blot was used to examine the expression of GRP78 and CHOP.Results：① Compared with the negative control,tunicamycin resulted in cardiomyocyte injury,which was time and concentration-dependent（P0.05,n=12）.② The treatment of tunicamycin produced 57.4±3.2 %（n=12） of cellular viability and 25.9±5.8%（n=3） of apoptotic population in cardiomyocytes by flow cytometry.③ The levels of GRP78 and CHOP upregulated at 6 h,respectively.After tunicamycin treatment for 24h,the upregulation of GRP78 and CHOP reached the maximum.Conclusions：Tunicamycin-induced apoptotic model in cultured neonatal rat cardiomyocytes were successfully constructed The optimal concentration and action time of tunicamycin treatment was 100ng/ml,72h,respectively.

关 键 词：衣霉素 内质网应激 凋亡 GRP78 CHOP 

分 类 号：Q95-3[生物学—动物学] R54[医药卫生—心血管疾病]