RNA干扰靶向抑制结缔组织生长因子对心肌纤维化的影响

The Effect of Targeting Inhibition of Connective Tissue Growth Factor on Myocardial Fibrosis by RNA Interference

背景心肌纤维化是心脏重塑的重要特征,结缔组织生长因子具有刺激成纤维细胞增殖及促进细胞外基质合成的生物学功能,是一种重要的促纤维化细胞因子,然而其对心肌纤维化的作用还没有得以系统研究。目的研究RNA干扰技术靶向抑制结缔组织生长因子(CTGF)对自发性高血压大鼠心肌组织纤维化指标的影响。方法20只自发性高血压大鼠随机分为未干预的SHR组和CTGF RNA干扰(RNAi)组,并设置WKY组大鼠为正常对照。在RNA干扰结束后,处死大鼠获取血液和心脏标本,计算左室质量/体质量(LVM/BM),ELISA法检测血浆纤维连接蛋白(FN)和层黏蛋白(LN)浓度;逆转录PCR和Western blot检测心肌组织CTGF及FN的mRNA和蛋白表达,免疫组化技术分析CTGF及FN的表达。天狼星红染色及羟脯氨酸测定分析心肌组织胶原类型及代谢水平。结果RNA干扰组血压水平和LVM/BM较SHR组无明显差异(P>0.05),RNA干扰使CTGF和FN mRNA表达分别下调60%和54%(P<0.01),蛋白表达分别下调30%和44%(P<0.01);免疫组化显示了同样的抑制效应,CTGF主要存在于心肌细胞,而FN主要在心肌间质表达。天狼星红染色显示RNA干扰组胶原沉积减少,偏光显微镜下观察到RNA干扰主要抑制了Ⅰ型胶原的合成;心肌组织羟脯氨酸含量和心肌组织胶原容积分数(CVF)在RNA干扰组分别显著减少36%和30%(P<0.01)。结论靶向抑制CTGF可以显著抑制细胞外基质在心肌间质的沉积,CTGF靶向性RNA干扰的抗心肌纤维化效应与降压无关。

Background Myocardial fibrosis is one of many features of cardiac remodeling. Connective tissue growth factor engendered profibrotic cytokine stimulating fibroblasts proliferation and promoting extracellular matrix deposition. Objeetive To investigate the impact on myocardial fibrosis by inhibition of connective tissue growth factor（CTGF） by RNA interference（RNAi） in spontaneous hypertension rat（SHR）. Methods Twenty SHR were randomly divided into SHR group and RNAi group, Wistar-Kyoto rats（n=8） were served as control. After RNA interference, rats were sacrificed and the blood and hearts were harvested. The LVM/BM ratio was caculated, and the plasma concentration of fibronectin（FN） and laminin（LN） were assessed by ELISA. The mRNA and protein of CTGF and FN in myocardium were detected by RT-PCR and Western Blotting, with the localization of CTGF and FN analyzed with immunohistochemistry technique. The collagen deposition were evaluated with 0.1% sirius-picric staining, and the hydroxyproline of myocardium were detected by colorimetry. Results There were no difference in blood pressure and LVM/BM between SHR and RNAi group（P〉0.05）. mRNA expression of CTGF and FN in myocardium in RNAi group were decreased markedly about 600/6o and 54% respectively（P〈0.01）, while protein expression reduced by 30% and 44% （P〈0.01）, which was confirmed by histological study. Immunohistochemistry showed the CTGF mainly localized in cardiomyocytes, whereas FN majorly in interstitium of myocardium. Myocardial volume fraction（CVF） and hydroxyproline were remarkably decreased 3 0 % and 3 6 % respectively in RNAi group （ P 〈 0. 0 1 ） . Conclusions Targeted inhibition of CTGF produced significant decrease of extracellular matrix deposition in myocardium. CTGF targeted RNA interference showed anti-fibrotic effect independent of lower the blood pressure. CTGF may be associated with the development and progress of myocardial fibrosis.