摘要
目的:探讨亲环素D(CypD)对棕榈酸诱导的小鼠胰岛β细胞株MIN6细胞凋亡的影响及机制。方法:在MIN6细胞中建立CypD过表达稳转细胞系与CypD敲低稳转细胞系,将细胞分为对照组(未转染病毒)、OE-ctrl组[转染过表达对照病毒未用棕榈酸(PA)处理]、OE-ctrl+PA组、OE-CypD组(转染CypD过表达病毒未用PA处理)、OE-CypD+PA组、KD-ctrl组(转染敲低对照病毒未用PA处理)、KD-ctrl+PA组、KD-CypD组(转染CypD敲低病毒未用PA处理)、KD-CypD+PA组,每组3个复孔。流式细胞仪检测各组细胞凋亡率,实时荧光定量聚合酶链反应与Western blot法检测各组CypD的mRNA及蛋白表达量。Western blot法检测凋亡相关蛋白[B细胞淋巴瘤2(Bax)、B细胞淋巴瘤2相关X蛋白(Bcl-2)、半胱氨酸依赖的天冬氨酸蛋白酶(Caspase)3、细胞色素c(Cyt-c)、凋亡酶激活因子1(Apaf-1)]的表达。共聚焦显微镜检测细胞内钙离子荧光。两组间比较采用t检验,多组间比较采用单因素方差分析。结果:与对照组相比,随着PA浓度上升,MIN6细胞凋亡率显著升高(P<0.01),CypD mRNA(分别为1.00±0.04和1.88±0.02)和CypD蛋白表达量(分别为0.049±0.004和0.577±0.006)均明显升高,差异具有统计学意义(P<0.01)。与OE-ctrl+PA组比较,OE-CypD+PA组细胞凋亡率升高[分别为(31.33±2.72)%和(24.95±0.94)%,P<0.05],细胞内钙离子荧光信号增加(分别为27.62±0.74和24.61±0.15,P<0.01),凋亡相关蛋白Bax、Caspase3、Cyt-c、Apaf-1水平升高(均P<0.01),细胞存活蛋白Bcl-2水平降低(P<0.01)。与KD-ctrl+PA组比较,KD-CypD+PA组细胞凋亡率降低[分别为(22.87±0.34)%和(26.91±0.93)%,P<0.05],细胞内钙离子荧光信号减少(分别为23.26±0.65和27.82±0.86,P<0.01),凋亡蛋白Bax、Caspase3、线粒体相关凋亡蛋白Cyt-c、Apaf-1水平降低(均P<0.01),Bcl-2水平升高(P<0.01)。结论:CypD加重PA诱导的MIN6细胞脂毒性损伤,可能与线粒体功能障碍有关。敲低CypD对脂毒性下的MIN6细胞有保护作用。
Objective To investigate the effect of cyclophilin D(CypD)on the apoptosis of mouse pancreatic isletβcell MIN6 induced by palmitic acid and its mechanism.Methods Stable CypD-overexpressing(OE)and CypD knockdown(KD)cell line was established in MIN6 cells by lentiviral transfection.The experiment wasgrouped as follows:control group[(untransfected virus without palmitic acid treatment(PA)],OE-control(ctrl)group,OE-ctrl+PA group,OE-CypD group(transfected CypD overexpression virus without PA treatment),OE-CypD+PA group,KD-ctrl group(transfected knockdown control virus without PA treatment),KD-ctrl+PA group,KD-CypD group(transfected with CypD knockdown virus without PA treatment),KD-CypD+PA group.Flow cytometry was used to detect the apoptosis rate of each group.The quantitative real-time polymerase chain reaction and Western blot were used to detect the effect of PA treatment on the expression of CypD.Western blot was used to detect the expression of apoptosis-related proteins Bcl-2 associated X protein(Bax),B cell lymphoma-2(Bcl-2),Caspase3,cytochrome c(Cyt-c),and apoptotic protease activating factor(Apaf-1).Intracellular calcium ion fluorescence was detected via confocal microscopy.The t-test was used for comparison between the two groups,and the one-way analysis of variance was used for the comparison among multiple groups.Results Compared with the control group,with the increase of PA concentration,the apoptosis rate of MIN6 cells increased significantly(P<0.01),CypD mRNA(1.00±0.04 vs.1.88±0.02,respectively)and CypD protein expression(0.049±0.004 vs.0.577±0.006,respectively)were significantly increased,and the difference was statistically significant(P<0.01).Compared with the OE-ctrl+PA group,the apoptosis rate of the OE-CypD+PA group increased[(31.33±2.72)%vs.(24.95±0.94)%,respectively,P<0.05],and the intracellular calcium ion fluorescence signal increased(27.62±0.74 vs.24.61±0.15,P<0.01),the levels of apoptosis-related proteins Bax,Caspase3,Cyt-c,and Apaf-1 increased(P<0.01),and the level of cell surviv
作者
程思源
吴玉文
何光珍
王华蔚
胡勇
徐焱成
Cheng Siyuan;Wu Yuwen;He Guangzhen;Wang Huawei;Hu Yong;Xu Yancheng(Department of Endocrinology,Zhongnan Hospital of Wuhan University,Wuhan 430071,China)
出处
《中华糖尿病杂志》
CAS
CSCD
北大核心
2021年第8期814-820,共7页
CHINESE JOURNAL OF DIABETES MELLITUS
基金
国家自然科学基金(81970718)。
关键词
亲环素类
胰岛Β细胞
脂毒性
线粒体通透性转换孔
Cyclophilins
Pancreatic isletβcells
Lipotoxicity
Mitochondrial permeability transition pores
