CAJ | 학술논문

目的探讨钙敏感受体(CaSR)对缺氧/复氧(A/R)所诱导乳鼠心肌细胞凋亡的影响及其机制.方法原代培养乳鼠心室肌细胞缺氧2 h/复氧24 h,建立A/R心肌细胞损伤模型.心肌细胞随机分为三组:对照组、A/R组和CaSR激动剂氯化钆(GdCl3)组.四氮唑嗅盐法(MTT)检测细胞的存活率,原位缺口末端标记法(TUNEL)分析细胞凋亡率,蛋白印迹法检测CaSR、半胱氨酸天冬酶(caspase)-3、9和细胞色素c(Cytc)蛋白的表达.结果 TUNEL染色显示A/R后心肌细胞凋亡为(17±3)%,与对照相比,心肌细胞凋亡明显增加,CaSR表达也增加.CaSR激动剂GdCl3使心肌细胞凋亡进一步增加至(28±4)%,MTT检测表明心肌细胞存活率仅为(51.2±6.8)%;CaSR、caspase-3、caspase-9和Cytc表达进一步上调,明显高于A/R组.结论 CaSR通过线粒体Cytc-caspase-3途径参与了A/R所诱导的心肌细胞凋亡.
목적 탐토개민감수체(CaSR)대결양/복양(A/R)소유도유서심기세포조망적영향급기궤제.방법 원대배양유서심실기세포결양2 h/복양24 h,건립A/R심기세포손상모형.심기세포수궤분위삼조:대조조、A/R조화CaSR격동제록화구(GdCl3)조.사담서후염법(MTT)검측세포적존활솔,원위결구말단표기법(TUNEL)분석세포조망솔,단백인적법검측CaSR、반광안산천동매(caspase)-3、9화세포색소c(Cytc)단백적표체.결과 TUNEL염색현시A/R후심기세포조망위(17±3)%,여대조상비,심기세포조망명현증가,CaSR표체야증가.CaSR격동제GdCl3사심기세포조망진일보증가지(28±4)%,MTT검측표명심기세포존활솔부위(51.2±6.8)%;CaSR、caspase-3、caspase-9화Cytc표체진일보상조,명현고우A/R조.결론 CaSR통과선립체Cytc-caspase-3도경삼여료A/R소유도적심기세포조망.
Objective Calcium-sensing receptor(CaSR)belongs to the family C of G-protein coupled receptors.This study was carried out to observe the influence and mechanism of CaSR on anoxia/reoxygenation(A/R)-induced cardiomyocytes apoptosis.Methods The model of A/R injury was established through anoxia for 2 hours and reoxygenation for 24 hours in cultured cardiomyocytes of neonatal rats.Cardiomyocytes were randomly divided into three groups:control group,A/R group and GdCl3 group(300μmol/L GdCl3 was added to the culture medium at the beginning of reoxygenation).Apoptosis of cardiomyocytes was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL).The expression of CaSR,cysteine-requiring aspartate protease(caspase)-3,9 and cytochrom c (Cytc)were analyzed by Western blot.Results The TUNEL showed that cardiomyocytes apoptosis was 17%±3% in A/R group,and was higher than that in the control group.At the same time,expression of CaSR in A/R group was markedly increased in response to control group.Compared with A/R group,GdCl3,a specific activator of CaSR,further enhanced cardiomyocytes apoptosis to (28±4)% and decreased the ability of cardiomyocytes to (51.2±6.8)%,along with an increment in CaSR,caspase-3,caspase-9 and Cytc expressions.Conclusion CaSR is involved in anoxia/reoxygenation-induced apoptosis of neonatal rat cardiomyocytes through Cytc-caspase-3 pathway.