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

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(P0.05);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(P0.05)。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。

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

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 (DMEMfor 48h); (2) PE group (PE, 50 uM, stimulated for 48 h); (3) Preconditioning groups: group A: Preconditioned with different PE concentration (preconditioned with PE 50 uM for 12 h, remove PE and cultured for 12 h); group B: Preconditioned for different preconditioning time with PE of 50 uM (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 uM for 48 h after the preconditioning treatment. Cell phenotype was determined by immunofluorescence staining of alpha-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 beta-MHC expression were significantly increased in PE group cells (P<0.05). Myocardial hypertrophy was relieved in preconditioning group cells, wherein group which stimulated with PE of 50 uM for twice 6 h the effect was most significant (P<0.05).Conclusion:Hypertrophic preconditioning can reduce the extent of cardiac hypertrophy induced by PE, exerting a protective effect on cardiomyocytes.