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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 investigate the effects of hypertrophic preconditioning on cardiomyocyte hypertrophy induced by phenylephrine (PE). Methods: Primary cultured neonatal SD rat cardiomyocytes were isolated and cultured with collagenase and differential adhesion method. The rats in control group were given routine culture for 48 h, PE group (50 μM PE for 48 h) Different pre-stimulation + PE groups: A, different concentrations of PE (10,20,50μM) pre-stimulation (12h intervention, 12h conventional culture); B, PE Intervention, 6 h conventional culture; Period-2, 6 h intervention, 6 h conventional culture, again 6 h intervention, 6 h conventional culture; Period-3,8 h intervention, 8 h conventional culture; , 12 h routine culture). After pre-stimulation, PE (50μM) was used to stimulate 48h. Immunofluorescence staining of α-actin and laser scanning confocal microscopy were used to observe the cell phenotype. Image J software was used to calculate the surface area of cardiomyocytes. The expression of HbA1c was detected by real-time quantitative PCR. Results: The purity of isolated cardiomyocytes was over 90%. Compared with the control group, the myocardial cells in PE group were significantly enlarged and the cell surface area increased 2.3-fold. The levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin (P <0.05). However, the hypertrophic phenotype of cardiomyocytes in different pre-stimulated + PE groups was relieved obviously. The pre-stimulation of PE (50μM) for two times was the most significant (P <0.05). Conclusion: Hypertrophic preconditioning can reduce the extent of PE-induced hypertrophy of cardiomyocytes and thus have a protective effect on cardiac hypertrophy.