促红细胞生成素促心肌细胞肥大的实验研究

The Effect of Erythropoietin on cardiac hypertrophy

目的观察促红细胞生成素(Erythropoietin,Epo)对离体乳鼠心肌细胞肥大形态学及分子生物学表现的影响。方法(1)新生大鼠原代心肌细胞分为空白对照、AngⅡ1×10^(-6)M和Epo10 U/ml干预组,取24 h、48 h和72 h三个时间点固定细胞,cTnI免疫组化染色,比较各组细胞面积;(2)RT-PCR法检测比较空白对照和Epo 10 U/ml干预30min c-fos mRNA和2 h ANP和BNP mRNA表达;(3)Western blot检测Epo10U/ml刺激p-ERK表达的时间变化。4)Western blot检测不同浓度Epo刺激p-ERK表达的差异。结果(1)AngⅡ和Epo刺激均使心肌细胞面积增大(P<0.05,24 h、48 h和72 h,AngⅡ组VS Con组;P<0.05,48 h和72 h,Epo组VS Con组);(2)Epo10 U/ml干预组心肌细胞30min c-fos mRNA表达量与对照组相比差异不明显,2h ANP mRNA和BNP mRNA表达量和对照组相比,明显升高(P<0.05);(3)Western blot结果显示,Epo干预心肌细胞后p-ERK2表达随时间出现先升高后下降的变化,30 min时达到高峰,与干预前和干预后120min差别具有统计学意义(P<0.05,30 min VS 0,120 min);(4)随Epo干预浓度增大,p-ERK2表达水平升高(P<0.05,Epo10 U/ml和Epo100 U/ml组VS Con组)。Epo刺激p-ERK1表达也出现相同的时间浓度趋势,但各组差别无统计学意义。结论Epo刺激可使心肌细胞表面积增大,促进肥大基因ANP、BNP mRNA表达的升高,并刺激p-ERK出现时间相关性和浓度依赖性表达。

Objective To observe the effect of Erythropoietin on cardiac hypertrophy. Methods （1） Primary cardiac myocytes were isolated from neonatal Sprague-Dawley rats and cultured. Three groups were included： control group,Ang II 1 × 10^-6M treated group and Epo 10 U/ml treated group. Culture cells were fixed at 24 h, 48 h and 72 h, and immunohistochemieal stained with cTnI. The sizes of the cardiomyocytes were measured with light microscopy. （2） Cardiomyocytes were divided into control group and the Epo 10 U/ml treated group. The total RNA of the eardiomyocytes were extracted and RT-PCR was introduced to semi-quantify c-fos mRNA expression after 30mins treated, similarly, ANP and BNP mRNA were measured after 2hs treated. （3） Total cellular protein was extracted and Western blot detection was Used to quantify the myocardial p-ERK with Epo 10U/ml treated for 0min （control group）, 10 min, 15 rain, 30 min, 60 min and 120 min respectively. （4）Epo treated cardiomyocytes for 30min, with the final concentration 0 （control group）, 5 U / ml, 10 U / ml and 100 U / ml respectively. Total cellular protein was extracted and myocardial p-ERK were detected by western blot and then compared. Results （ 1 ） After 24 hours exposure, the mean size of the cells were significantly increased for Ang II treated and Epo treated group （ P 〈 0.05, 24 h, 48 h and 72 h, Ang II group VS Control group;P 〈0.05, 48h and 72 h,Epo group VS Control group）. （2） With Epo10U/ml intervention in cardiac myocytes, ANP and BNP mRNA expression were significantly higher than that of control group（P 〈 0.05, Epo treated group VS control group）, yet c-fos mRNA expression was not increased significantly. （3） p-ERK2 expression in cardiac myocytes first increases then declines within the 120 min treated time with Epo 10 U/ml intervention. The expression level was at peak for 30 min treated group （ P 〈 0.05, 30 min treated group VS 0,120 rain treated groups）. （4） Increasing concentration of Epo intervention resulted in increased p-ERK2 expression levels in cardiac myocytes, p-ERK2 expression was significantly higher in the groups of Epo10 U/ml and 100 U/rrd treated than that of the control group （P 〈 0.05, Epo10 U/ml group and Epo100 U/ml group VS Control group）. The p-ERK1 expression was also changed as p-ERK2, yet the differences were not significant. Conclusion Addition of Epo can induce enlargement of cardiomyocytes. Epo can stimulate the expression of ANP and BNP genes and induce ERK activation in a time- and concentration-dependent manner.