缬沙坦对心肌成纤维细胞转化生长因子和结缔组织生长因子表达的影响

Effect of valsartan on TGF-β and CTGF expression in myocardial fibroblast

目的通过体外培养新生大鼠心肌成纤维细胞研究缬沙坦对转化生长因子β(transforming growthfactorβ,TGF-β)和结缔组织生长因子(connective tissue growth factor,CTGF)表达的影响。方法胶原酶消化法分离心肌成纤维细胞(cardiac fibroblasts,CFs),构建AngⅡ诱导CFs的心肌纤维化细胞模型。各观察点以不同浓度的缬沙坦干预模型,光镜下观察细胞生长状况。采用MTT比色法检测缬沙坦对CFs增殖的影响,AO/EB染色法分析CFs活力,电镜下研究亚细胞结构;采用反转录聚合酶链反应(RT-PCR)技术测定CFsTGF-β1及CTGF mRNA的表达。WesternBlot法测定TGF-β1、CTGF蛋白的表达。结果光镜下可见:CFs于种植24h后完成贴壁,AngII干预后的细胞显著增殖,不同浓度的缬沙坦处理后的细胞24h内均表现为凋亡。MTT及AO/EB染色表明缬沙坦浓度为10μmol/L干预8h抑制增殖效应最为显著。AO/EB染色及电镜结果提示其作用机制为诱导细胞凋亡。RT-PCR及Western Blot结果显示,上述条件下,CFs中TGF-β和CTGF表达明显下调。结论缬沙坦浓度为10μmol/L作用8h后CFs凋亡率最高,其作用机制与TGF-β及CTGF的表达下调有关。

Objective To culture cardiac fibroblasts（CFs） in Vitro, and investigate the effect of valsartan on the expression of transforming growth factor β （TGF-β）and connective tissue growth factor （CTGF）. Methods CFs were isolated with eollagenase digestion. In order to construct the myocardial fibroblast model, angiotensin Ⅱ （Ang Ⅱ） was added to the culture medium. The model cells were divided into several groups,and different concentrations of valsartan were added in. The CFs growth condition was observed and recorded by light microscope at different time after intervention. The viability of CFs was quantitatively assessed by MTT assay and AO/EB staining. Transmission electron microscopy was used to observe the subcellular structure. The expression of TGF-β and CTGF mRNA and protein were detected by reverse transcription polymerase chain reaction （RT-PCR） and Western blot respectively. Results The CFs finished adhering and growing 24 hours after plantation. The growth rate of CFs was increased significantly after AnglI was added in. Being intervened by different concentrations of valsartan, characteristic morphological alteration of apoptosis could be seen within 24 hours in parts of the CFs. MTT assay and AO/EB staining showed that valsartan at a concentration of 10/μmol/L had the best inhibitive effect on CFs growth after 8 hours. AO/EB staining and transmission electron microscopy suggested that CFs apoptosis was the main cause of death. It is proved that, under this condition, TGF-β and CTGF expression of CFs was significantly decreased by RT-PCR and Western Blot. Conclusion The apoptosis rate of CFs was the highest while valsartan at a concentration of 10μmol/L intervening the cells 8 hours, which may be related to the down regulation expression of TGF-β and CTGF.