CAJ | 학술논문

目的探讨葡萄糖调节蛋白78 (GRP78)在七氟醚预处理抑制大鼠心肌细胞凋亡中的作用.方法采用随机数字表法将培养的大鼠心肌细胞分5组(n=30),正常对照组(C组):细胞不经任何处理;缺氧复氧组(H/R组):细胞在95％N2-5％CO2混合气体中缺氧2h后放回95％空气-5％CO237℃培养箱内复氧lh;七氟醚预处理组(S组):细胞经2.5％七氟醚孵育20 min,洗脱10 min后进行缺氧复氧;siRNA-GRP78组:采用siRNA-GRP78 100 nmol/L转染心肌细胞,24 h后进行七氟醚预处理及缺氧复氧处理;siRNA对照组(siRNA组):采用随机序列核酸siRNA转染心肌细胞,其它处理同siRNA-GRP78组.各组处理结束后,采用Western blot法测定心肌细胞GRP78、胞浆和线粒体细胞色素c的表达;采用ELISA法测定培养液LDH和CK的活性;采用流式细胞仪测定细胞凋亡率;采用Fura-2/AM钙离子荧光探针法测定细胞内游离Ca2+浓度([Ca2+]i);采用荧光分光光度法测定线粒体膜通透性转运孔(mPTP)开放程度.结果与C组比较,H/R组心肌细胞GRP78和胞浆细胞色素c表达上调,培养液LDH和CK活性、细胞凋亡率、[Ca2+]i升高,线粒体细胞色素c表达下调(P＜0.01);与H/R组比较,S组心肌细胞GRP78和线粒体细胞色素c表达上调,培养液LDH和CK活性、细胞凋亡率、[Ca2+]i和mPTP开放程度降低,胞浆细胞色素c表达下调(P＜0.01),siRNA组上述指标差异无统计学意义(P＞0.05);与S组比较,siRNA-GRP78组心肌细胞GRP78和线粒体细胞色素c表达下调,培养液LDH和CK活性、细胞凋亡率、[Ca2+]i和mPTP开放程度升高,胞浆细胞色素c表达上调(P＜0.01).结论 GRP78参与了七氟醚预处理抑制大鼠心肌细胞凋亡的作用,机制与维持细胞内Ca2+稳态,抑制mPTP开放有关.
목적 탐토포도당조절단백78 (GRP78)재칠불미예처리억제대서심기세포조망중적작용.방법 채용수궤수자표법장배양적대서심기세포분5조(n=30),정상대조조(C조):세포불경임하처리;결양복양조(H/R조):세포재95％N2-5％CO2혼합기체중결양2h후방회95％공기-5％CO237℃배양상내복양lh;칠불미예처리조(S조):세포경2.5％칠불미부육20 min,세탈10 min후진행결양복양;siRNA-GRP78조:채용siRNA-GRP78 100 nmol/L전염심기세포,24 h후진행칠불미예처리급결양복양처리;siRNA대조조(siRNA조):채용수궤서렬핵산siRNA전염심기세포,기타처리동siRNA-GRP78조.각조처리결속후,채용Western blot법측정심기세포GRP78、포장화선립체세포색소c적표체;채용ELISA법측정배양액LDH화CK적활성;채용류식세포의측정세포조망솔;채용Fura-2/AM개리자형광탐침법측정세포내유리Ca2+농도([Ca2+]i);채용형광분광광도법측정선립체막통투성전운공(mPTP)개방정도.결과 여C조비교,H/R조심기세포GRP78화포장세포색소c표체상조,배양액LDH화CK활성、세포조망솔、[Ca2+]i승고,선립체세포색소c표체하조(P＜0.01);여H/R조비교,S조심기세포GRP78화선립체세포색소c표체상조,배양액LDH화CK활성、세포조망솔、[Ca2+]i화mPTP개방정도강저,포장세포색소c표체하조(P＜0.01),siRNA조상술지표차이무통계학의의(P＞0.05);여S조비교,siRNA-GRP78조심기세포GRP78화선립체세포색소c표체하조,배양액LDH화CK활성、세포조망솔、[Ca2+]i화mPTP개방정도승고,포장세포색소c표체상조(P＜0.01).결론 GRP78삼여료칠불미예처리억제대서심기세포조망적작용,궤제여유지세포내Ca2+은태,억제mPTP개방유관.
Objective To investigate the role of glucose-regulated protein 78 (GRP78) in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats.Methods The cultured cardiomyocytes of Sprague-Dawley rats were randomly divided into 5 groups (n =30 each) using a random number table:control group (group C),hypoxia-reoxgenation (H/R) group,sevoflurane preconditioning group (group S),siRNA-GRP78 group and siRNA control group.H/R was produced by 2 h exposure of cells to 95％ N2-5％ CO2 in an air-tight chamber at 37 ℃,followed by reoxygenation with 95％ O2-5％ CO2 in an air-tight chamber at 37 ℃ for 1 h.In group S,the cells were incubated with 2.5％ sevoflurane for 20 min,followed by 10-min washout before H/R.In siRNA-GRP78 group,the cells were transfected with siRNA-GRP78 100 nmol/L,and 24 h later preconditioning with sevoflurane was performed and H/R was produced.In siRNA group,cells were transfected with siRNA,and the other treatments were similar to those previously described in siRNA-GRP78 group.After treatment in each group,the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm and mitochondria was detected by Western blot.Lactic dehydrogenase (LDH) and creatine kinase (CK) activities in the culture medium of each group were determined by ELISA.The apoptosis in myocardial cells was assessed by flow cytometry.Apoptotic rate was calculated.Intracellular free Ca2+ concentration ([Ca2+]i) was measured with the fluorescent probe Fura-2/ AM.The opening of mPTP was measured by fluorescence spectrophotometry.Results Compared to group C,the expression of GRP78 in myocardial cells and cytochrome c in cytoplasm was significantly up-regulated,LDH and CK activities in the culture medium,apoptotic rate and [Ca2+]i were increased,and the expression of cytochrome c in mitochondria was down-regulated in H/R group.Compared to group H/R,the expression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly up-regulated,LDH and CK activities in the culture medium,apoptotic rate,[Ca2+] i and opening of mPTP were decreased,and the expression of cytochrome c in cytoplasm was down-regulated in group S,and no significant change was found in the parameters mentioned above in siRNA group.Compared to group S,the expression of GRP78 in myocardial cells and cytochrome c in mitochondria was significantly down-regulated,LDH and CK activities in the culture medium,apoptotic rate,[Ca2+] i and opening of mPTP were increased,and the expression of cytochrome c in cytoplasm was up-regulated in group siRNA-GRP78.Conclusion GRP78 is involved in sevoflurane preconditioning-induced inhibition of apoptosis in cardiomyocytes of rats,and the mechanism is related to maintenance of intracellular Ca2+ stability and inhibition of opening of mPTP.