CAJ | 학술논문

目的：以蛋白激酶C-ε(PKC-ε)转位为切入点,探讨2类多巴胺受体(DR2)在心肌缺血后适应抑制细胞凋亡中的作用及可能机制。方法复制原代培养乳鼠心肌细胞缺氧/复氧和缺血后适应模型。 MTT检测心肌细胞的存活率；Hoechst 33342染色观察细胞凋亡；Western blotting检测Bcl-2、caspase-3、caspase-9、PKC-ε蛋白的表达和细胞色素C( Cyt c)的释放；免疫共沉淀检测PKC-ε和DR2的相互作用。结果与正常组比较,缺氧/复氧组细胞存活率降低,细胞凋亡增加,促凋亡因子( Cyt c、caspase-3、caspase-9)表达增加,抑凋亡因子(Bcl-2)表达亦增加,PKC-ε没有转位到细胞膜,PKC-ε和DR2不存在相互作用。与缺氧/复氧组比较,缺血后适应明显升高细胞存活率,降低细胞凋亡,抑制促凋亡因子(Cyt c、caspase-3、caspase-9)表达,促进抑凋亡因子(Bcl-2)表达,PKC-ε转位到细胞膜,PKC-ε和DR2存在相互作用。与缺血后适应组比较,DR2激动剂进一步增加缺血后适应的保护作用,而DR2抑制剂则取消了DR2激动剂的作用。结论 DR2参与心肌缺血后适应抑制细胞凋亡,其机制与DR2促进PKC-ε转位及DR2和PKC-ε存在相互作用有关。
목적：이단백격매C-ε(PKC-ε)전위위절입점,탐토2류다파알수체(DR2)재심기결혈후괄응억제세포조망중적작용급가능궤제。방법복제원대배양유서심기세포결양/복양화결혈후괄응모형。 MTT검측심기세포적존활솔；Hoechst 33342염색관찰세포조망；Western blotting검측Bcl-2、caspase-3、caspase-9、PKC-ε단백적표체화세포색소C( Cyt c)적석방；면역공침정검측PKC-ε화DR2적상호작용。결과여정상조비교,결양/복양조세포존활솔강저,세포조망증가,촉조망인자( Cyt c、caspase-3、caspase-9)표체증가,억조망인자(Bcl-2)표체역증가,PKC-ε몰유전위도세포막,PKC-ε화DR2불존재상호작용。여결양/복양조비교,결혈후괄응명현승고세포존활솔,강저세포조망,억제촉조망인자(Cyt c、caspase-3、caspase-9)표체,촉진억조망인자(Bcl-2)표체,PKC-ε전위도세포막,PKC-ε화DR2존재상호작용。여결혈후괄응조비교,DR2격동제진일보증가결혈후괄응적보호작용,이DR2억제제칙취소료DR2격동제적작용。결론 DR2삼여심기결혈후괄응억제세포조망,기궤제여DR2촉진PKC-ε전위급DR2화PKC-ε존재상호작용유관。
Objective To investigate the effect and possible mechanism of dopamine receptor-2 (DR2) activation in ischemic postconditioning ( PC) inhibited cardiomyocytes apoptosis through translocation of PKC-ε. Method The hypoxia/reoxygenation ( H/R) injury and PC models was established using primarily cultured neonatal rat cardio-myocytes. The cell survival rate was detected by MTT. The cell apoptosis was observed using Hoechst 33342 sta-ning. The protein expression of Bcl-2, caspase-3, caspase-9, the release of cytochrome c( Cyt c) and translocation of PKC-εwere analyzed using Western blotting. The interaction of DR2 and PKC-εwas tested by co-immunoprecipi-tation. Result Compared with Control group, the cell survival rate was decreased, cardiomyocytes apoptosis was heightened, the expression of Bcl-2,caspase-3,caspase-9 and the release of Cyt c were increased, PKC-ε were not translocated to the cell membrane and the interaction of DR2 and PKC-ε was not exist in H/R group. Compared with H/R group, PC increased the cell survival rate, decreased cardiomyocytes apoptosis, down-regulated the ex-pression of caspase-3, caspase-9 and the release of Cyt c, up-regulated the expression of Bcl-2, promoted transloca-tion of PKC-ε and the interaction of DR2 and PKC-ε. Compared with PC group, the agonist of DR2 further in-creased cardioprotection of PC, however, the antagonist of DR2 canceled this effect of DR2 agonist. Conclusion DR2 activation is involved in PC inhibited apoptosis by promoting translocation of PKC-ε.