不同浓度胰岛素对体外培养新生鼠心肌细胞生长和增殖的影响

Different concentrations of insulin affect the growth and proliferation of in vitro cultured cardiomyocytes from neonatal rats

背景:小鼠高胰岛素血症模型研究发现,高胰岛素可引起心肌肥厚的发生。目的:观察胰岛素对体外培养的新生鼠心肌细胞增殖和肥大的浓度效应。设计、时间及地点:细胞学体外观察,于2006-01/2007-03在新乡医学院解剖学与组织胚胎学重点学科实验室完成。材料:1～3d龄Wistar新生鼠20只,胰岛素为美国sigma公司产品。方法:采用胰蛋白酶消化法和差速贴壁法体外分离培养和纯化新生鼠心室肌细胞,分别加入10-8,10-7,10-6mol/L胰岛素处理48h,并设立空白对照组,细胞周期分析选取10-7mol/L胰岛素组。主要观察指标:显微测量细胞直径、细胞活力和细胞计数,免疫细胞化学染色观察心肌细胞中细胞周期素E的变化。结果:各组心肌细胞活力均在90%以上。与空白对照组比较,10-8,10-7,10-6mol/L胰岛素组心肌细胞长径和短径均显著增加(t=2.7815,P<0.01);10-8,10-7mol/L胰岛素组心肌细胞数目无明显变化(P>0.05),10-6mol/L胰岛素组心肌细胞数目显著下降(t=1.9762,P<0.05);10-7mol/L胰岛素组心肌细胞内细胞周期素E阳性颗粒吸光度值明显下降(t=2.1743,P<0.05),平均灰度值明显增加(t=2.4587,P<0.05)。结论:10-8～10-6mol/L胰岛素具有促进新生鼠心肌细胞肥大的作用,但无法促进心肌细胞增殖。

BACKGROUND： Study on mouse models of hyperinsulinemia confirms that hyperinsulinemia can induce the occurrence of myocardial hypertrophy. OBJECTIVE： To study the effect of insulin on proliferation and hypertrophy of cardiomyocytes in neonatal mice in vitro cultured. DESIGN, TIME AND SETTING： The in vitro cytology experiment was performed at the Key Laboratory of Anatomy, Histology and Embryology of Xinxiang Medical University from January 2006 to March 2007. MATERIALS： Twenty Wistar neonatal mice aged 1-3 days were used in this study. Insulin was obtained from Sigma, USA. METHODS： Cardiomyocytes of neonatal mice were in vitro harvested, culture and purified by trypsin digestion and differential attachment technique. Cardiomyocytes were incubated in 10^-8, 10^-7, 10^-6 mol/L insulin for 48 hours. Blank control group was set up. 10^-7 mol/L insulin group was used for cell cycle analysis. MAIN OUTCOME MEASURES： Cell diameter, activity and counting were measured under a microscope. The expression of cyclin E in cardiomyocytes was measured using immunocytochemistry. RESULTS：Cardiomyocyte activity in each group was over 90%. Compared with the blank control group, long diameter and short diameter of cardiomyocytes in the 10^-8, 10^-7, 10^-6 mol/L insulin groups were significantly increased （t=2.781 5, P 〈 0.01）. The number of cardiomyocytes had no significant changes in the 10^-8, 10^-7 mol/L insulin groups （P 〉 0.05）, but the number of cardiomyocytes in the 10^-6 mol/L insulin group significantly decreased （t=1.976 2, P 〈 0.05）. The absorbance （A） of cyclin E positive granules in the cardiomyocytes significantly reduced in the 10^-7 mol/L insulin group （t=2.174 3, P 〈 0.05）, but mean gray value significantly increased （t=2.458 7, P 〈 0.05）. CONCLUSION： 10^-8-10^-6 mol/L insulin cannot promote the proliferation of cardiomyocytes, but can increase the hypertrophy in neonatal mice.