CAJ | 학술논문

目的观察内皮素-1(ET-1)诱导的心肌细胞肥大模型中细胞外信号调节激酶(ERK1/2)、磷酸化细胞外信号调节激酶(p-ERK1/2)、低氧诱导因子(HIF-1α),α-烯醇化酶(α-enolase)蛋白表达情况,探讨肥大心肌细胞α-enolase高表达的调控机制. 方法建立ET-1诱导的心肌细胞肥大模型,从细胞表面积、细胞蛋白合成速率和肌原纤维的重排3方面进行验证;将原代培养心肌细胞随机分为4组:(1)对照组;(2)PD98059干预组;(3)ET-1刺激组;(4)PD98059+ET-1刺激组.免疫印迹方法检测ERK1/2、p-ERK1/2、HIF-1α、α-enolase的蛋白表达. 结果 ET-1刺激后心肌细胞表面积为(1350.7±107.5)μm2,较对照组(896.1±70.2)μm2增加(P<0.05);ET-1刺激后心肌细胞[3H]亮氨酸掺入量较对照组增加[分别为(1387.9±14.8)dpm和(787.7±10.2)dpm,P<0.013;肌原纤维染色可见ET-1刺激屙心肌细胞肌原纤维排列较对照组紧密、染色浓,表明ET-1能诱导心肌细胞肥大.ERK1/2抑制剂PD98059预处理的心肌细胞在ET-1刺激后细胞表面积和细胞的[3H]亮氨酸掺入量均较ET-1刺激组减少[细胞表面积分别为(907.0±92.5)μm2和(1350.7±107.5)μm2,P<0.05;[3H]亮氨酸掺入量:(841.5±10.5)dpm和(1387.9±14.8)dpm,P<0.05].ET-1刺激后心肌细胞有p-ERK1/2表达,其抑制剂PD98059在抑制ERK1/2活化的同时也部分抑制了HIF-1α、α-enolase蛋白的表达. 结论 ERK1/2的活化与ET-1诱导的细胞肥大关系密切,在ET-1促心肌细胞肥大过程中,从MAPK/ERK1/2至HlF-1α及α-enolase这条信号通路町能参与α-enolase高表达的调控.
목적 관찰내피소-1(ET-1)유도적심기세포비대모형중세포외신호조절격매(ERK1/2)、린산화세포외신호조절격매(p-ERK1/2)、저양유도인자(HIF-1α),α-희순화매(α-enolase)단백표체정황,탐토비대심기세포α-enolase고표체적조공궤제. 방법 건립ET-1유도적심기세포비대모형,종세포표면적、세포단백합성속솔화기원섬유적중배3방면진행험증;장원대배양심기세포수궤분위4조:(1)대조조;(2)PD98059간예조;(3)ET-1자격조;(4)PD98059+ET-1자격조.면역인적방법 검측ERK1/2、p-ERK1/2、HIF-1α、α-enolase적단백표체. 결과 ET-1자격후심기세포표면적위(1350.7±107.5)μm2,교대조조(896.1±70.2)μm2증가(P<0.05);ET-1자격후심기세포[3H]량안산참입량교대조조증가[분별위(1387.9±14.8)dpm화(787.7±10.2)dpm,P<0.013;기원섬유염색가견ET-1자격아심기세포기원섬유배렬교대조조긴밀、염색농,표명ET-1능유도심기세포비대.ERK1/2억제제PD98059예처리적심기세포재ET-1자격후세포표면적화세포적[3H]량안산참입량균교ET-1자격조감소[세포표면적분별위(907.0±92.5)μm2화(1350.7±107.5)μm2,P<0.05;[3H]량안산참입량:(841.5±10.5)dpm화(1387.9±14.8)dpm,P<0.05].ET-1자격후심기세포유p-ERK1/2표체,기억제제PD98059재억제ERK1/2활화적동시야부분억제료HIF-1α、α-enolase단백적표체. 결론 ERK1/2적활화여ET-1유도적세포비대관계밀절,재ET-1촉심기세포비대과정중,종MAPK/ERK1/2지HlF-1α급α-enolase저조신호통로정능삼여α-enolase고표체적조공.
Objective To investigate the protein expression of ERK1/2,p-ERK1/2,HIF-1α and α-enolase in hypertrophic cardiomyocytes induced by ET-1 and explore the regulation mechanism of overexpression of α-enolase in hypertrophic cardiomyocytes. Methods ET-1-induced abnormal cardiomyocytes were used as model of cardiac hypertrophy. Cell surface area, [<'3>H]-leucine incorporation and the actin staining were measured to determine the extent of hypertrophy. Cultured cardiomyocytes were divided into 4 groups at random, control group, PD98059 treated group, ET-1 treated group and PD980594- ET-1 treated group. The protein expressions of ERK1/2, p-ERK1/2,HIF-1α and α-enolase were detected by immunoblotting analysis. Results Compared with the control group , cell surface area and [<'3>H] leucine incorporation were increased in ET-1 treated group ((1350.7±107.5)μtm<'2> vs. (896.1±70. 2)μtm<'2> , P<0.05; (1387.9±14.8) dpm vs. (787.7±10.2)dpm,P<0.013. Actin staining showed that ET-1-treated cardiomyoeytes had more intense actin staining and clear cross-striations than did control group, which suggested that myocardial cell hypertrophy could be induced by ET-1 in WKY neonatal cardiomyocytes. After MEK 1/2 inhibitor PD98059 was used, the cell surface area and [<'3>H] leucine incorporation were decreased in PD980594-ET-1 treated group compared with ET-1 treated group[(907.0±92.5)μm<'2> vs. (1350.7±107.5)μm<'2>;(841.5±10. 5)dpm vs. (1387.9±14.8)dpm, both P<0.05], which suggested that myocardial cell hypertrophy could be regulated by ERK1/2 signal pathway. Immunoblotting analysis showed that the protein expressions of p-ERK1/2, HIF-1α and α-enolase increased after ET-1 treatment, while PD98059 as an inhibitor of the upstream kinase of ERK1/2 was used, the protein expressions of HIF-1α and α-enolase were partially inhibited. Conclusions ET-1 induces hypertrophic cardiomyocytes through ERK1/2 phosphorylation in cultured neonatal rat cardiac myocytes. ERK1/2 and HIF-1α signal pathway may play an important role in the overexpression of α-enolase in the hypertrophic cardiomyocytes.