肥厚预刺激对苯肾上腺素诱导的心肌细胞肥大的保护作用

Hypertrophic Preconditioning can Attenuate Phenylephrine-induced Hypertrophy of Neonatal Rat Ventricular Cardiomyocytes in Vitro

目的:研究不同肥厚预刺激对苯肾上腺素(Phenylephrine,PE)诱导的心肌细胞肥大的影响。方法:胶原酶联合差速贴壁法分离培养原代SD乳鼠心肌细胞后分组:(1)对照组(常规培养48 h);(2)PE组(50μM PE刺激48 h);(3)不同预刺激+PE组:A,不同浓度的PE(10、20、50μM)预刺激(12 h干预,12 h常规培养);B,PE(50μM)预刺激不同时间(period-1,6 h干预,6 h常规培养;period-2,6 h干预,6 h常规培养,再次6 h干预,6 h常规培养;period-3,8 h干预,8 h常规培养;period-4,12 h干预,12 h常规培养)。预刺激后再用PE(50μM)刺激48 h。经细胞骨架蛋白(α-actining)免疫荧光染色,利用激光共聚焦显微镜观察细胞表型,Image J软件计算心肌细胞表面积,利用实时定量PCR检测肥厚相关标志物表达水平。结果:分离的心肌细胞纯度在90%以上。PE组较对照组心肌细胞明显肥大,细胞表面积增加2.3倍,心肌肥厚标记基因心钠肽(atrial natriuretic peptide,ANP)、脑钠肽(brain natriuretic peptide,BNP)和β肌球蛋白重链(βmyosin heavy chain,βMHC)表达明显升高(P<0.05);而不同预刺激+PE组心肌细胞肥大表型明显缓解,其中PE(50μM)两次6 h预刺激最为显著(P<0.05)。结论:肥厚预刺激可以减轻PE诱导的心肌细胞肥大的程度,从而对心肌肥厚有保护作用。

Objective： To study the protective effect of pre-stimulation on phenylephrine-induced cardiomyocyte hypertrophy.Methods： Collagenase and differential velocity adherent method was used to isolate neonatal SD rat ventricular cardiomyocytes（NRVCs）.The cultured NRVCs were grouped as followed：（1） Control group（DMEM for 48h）;（2） PE group（PE, 50 μM, stimulated for 48 h）;（3）Preconditioning groups： group A： Preconditioned with different PE concentration（preconditioned with PE 50 μM for 12 h, remove PE and cultured for 12 h）; group B： Preconditioned for different preconditioning time with PE of 50 μM（period-1. Preconditioned with PE for 6 h, remove PE and cultured for 6 h. period-2. Preconditioned for 6 h, conventional cultured for 6 h, and repeat. period-3. Preconditioned for 8 h, conventional cultured for 8 h. period-4. Preconditioned for 12 h, conventional cultured for 12 h）. The preconditioning group cells were stimulated with PE of 50 μM for 48 h after the preconditioning treatment. Cell phenotype was determined by immunofluorescence staining of α-actining and confocal microscopy. Image J software was applied to calculates the cell area. Real-time quantitative PCR was used to determine expression levels of hypertrophy markers genes. Results： The purity of isolated cardiomyocytes was above 90%. Surface area of PE group cells were about 2.3 times than that of control group cells, which shows sign of more severe hypertrophy. Marker genes myocardial hypertrophy, including ANP, BNP β-MHC expression were significantly increased in PE group cells（P0.05）. Myocardial hypertrophy was relieved in preconditioning group cells, wherein group which stimulated with PE of 50 μM for twice 6 h the effect was most significant（P0.05）. Conclusions： Hypertrophic preconditioning can reduce the extent of cardiac hypertrophy induced by PE, exerting a protective effect on cardiomyocytes.