体外模拟局部肾素血管紧张素系统对心肌纤维化的影响

Effects of local renin-angiotensin system on cardiac fibrosis in vitro

目的建立局部肾素血管紧张素系统(RAS)对大鼠心肌纤维化影响的体外模型。方法原代培养新生大鼠心肌细胞和心肌成纤维细胞,用RT-PCR鉴定两种细胞RAS各组分在基因水平的表达。从RAS各组分蛋白代谢的角度,采用依那普利阻断心肌细胞血管紧张素转换酶(ACE),放免法观察培养基中血管紧张素Ⅱ(AngⅡ)量的变化,结合Western blotting验证ACE2的存在,确定心肌细胞局部RAS各组分在蛋白水平的存在。以Transwell共培养心肌细胞和心肌成纤维细胞,构建局部RAS影响心肌纤维化的体外模型。结果在心肌细胞中检测到RAS各组分基因的表达,但在成纤维细胞中未发现肾素和ACE2的表达;对心肌细胞采用依那普利干预后AngⅡ呈剂量依赖性下降,10-7mol/L组[(36.24±3.27)pg/mL]与对照组[(44.08±0.76)pg/mL]有显著差异(P<0.05);Western blotting证实心肌细胞在蛋白水平有ACE2的表达。结论心肌细胞能够单独形成局部RAS;而成纤维细胞因为不能生成肾素和ACE2,则本身不能形成局部RAS。以Transwell体外共培养心肌细胞和心肌成纤维细胞能够建立一个用于研究局部RAS对心肌纤维化影响的体外模型。

Objective To study the feasibility of building the rat model of local renin-angiotensin system （RAS） on cardiac fibrosis in vitro. Methods The rat neonatal cardiomyocytes and the cardiac fibroblast cells were cultured in vitro, then RT-PCR were performed to identify the existence of different components of RAS in these two kinds of cells. Angiotensin-converting enzyme （ACE） in cardiomyocytes was blocked by Enalapril. The concentration of Angiotensin IX in the medium was detected with radio-immunological technology. The protein of ACE2 and different components of RAS were identified with Western blotting. Co-culturing the cardiomyocytes and the cardiac fibroblast cells in the Transwell system was performed to build the model of the affection on cardiac fibrosis produced by local RAS in vitro. Results The genes of RAS components were detected in the cardiomyocyte with RT-PCR. While in the fibroblast cell, renin and ACE2 were not found. AngⅡ dose-dependently decreased with Enalapril in cadiomyocytes. The concentration of AngⅡ of 10^-7 mol/L Enalapril group [ （36.24 ± 3.27） pg/mL] was much higher than that of the control group [ （44.08 ± 0.76） pg/ml ] （ P 〈 0.05）. ACE2 was detected positive by Western blotting. Conclusion Cardiomyocytes can form local RAS alone, while fibroblasts can not form local RAS with absence of renin and ACE2. Co-culturing the cardiomyocytes and cardiac fibroblast cells in vitro may be a good model to study the relationship between local RAS and cardiac fibrosis.