摘要
目的:探讨microRNA-494(miR-494)对转化生长因子β1(TGF-β1)刺激后大鼠肾间质成纤维细胞(NRK-49F)细胞外基质分泌的调控作用。方法:体外培养NRK-49F细胞株,并分为以下6组:TGF-β1(3ng/m L)组、miR-494模拟物(50ng/m L)组、miR-494抑制物(50ng/mL)组、miR-494模拟物(50ng/m L)+TGF-β1(3ng/m L)组、miR-494抑制物(50ng/m L)+TGF-β1(3ng/m L)组、空白对照组。茎环实时荧光定量PCR(RTqPCR)检测细胞miR-494、胶原蛋白I(ColI)、纤维连接蛋白(FN)mRNA;WesternBlot法检测细胞ColI蛋白。结果:与空白对照组比较,TGF-β1组miR-494(P<0.01)、ColI mRNA(P<0.05)及FN(P<0.01)mRNA表达显著升高;miR-494模拟物组ColI、FN mRNA表达显著升高(P<0.05);miR-494抑制物组ColI(P<0.05)、FN(P<0.01)mRNA表达显著降低。与TGF-β1组比较,miR-494模拟物+TGF-β1组ColI、FN mRNA表达显著升高(P<0.05);mi R-494抑制物+TGF-β1组ColI、FN mRNA表达显著降低(P<0.05)。各组间ColI蛋白与ColI mRNA的表达水平相一致。结论:miR-494可能参与调控并促进TGF-β1诱导肾间质成纤维细胞的细胞外基质分泌。
Objective： To explore the adjusting and controlling effect of microRNA-494（miR-494） on extracellular matrix of the secretion cells in renal interstitial fibroblast（NRK-49F） cells after stimulation of TGF-β1. Methods： NRK-49 F cells were cultured in vitro and divided into： TGF-β1（3 ng/m L） group, miR-494 mimics（50 ng/m L） group, miR-494 inhibitor（50 ng/m L） group, co-treated with miR-494 mimics（50 ng/m L） and TGF-β1（3 ng/m L） group, co-treated with miR-494 inhibitor（50 ng/m L） and TGF-β1（3 ng/m L） group, blank control group. The expression of mi R-494, collagen I（Col I）, fibronectin（FN） m RNAs were analyzed by stem-loop real-time quantitative PCR（RT-q PCR） techniques. The expression of Col I protein was analyzed by Western Blot. Results： Compare with blank control group, the expression of mi R-494（P〈0.01）, Col I（P〈0.05） and FN（P〈0.01） m RNA were significantly increased in TGF-β1 group, the expression of Col I and FN m RNA were significantly increased in mi R-494 mimics group（P〈0.05）, the expression of Col I（P〈0.05） and FN（P〈0.01） m RNAs were significantly decreased in mi R-494 inhibitor group. Compared with TGF-β1 group, the expression of Col I and FN m RNAs were significantly increased in co-treated with mi R-494 mimics and TGF-β1 group（P〈0.05）, the expression of ColI and FN mRNA were significantly decreased in co-treated with miR-494 inhibitor and TGF-β1 group（P〈0.05）, the expression of Col I protein was consistent with Col I mRNA（P〈0.05）. Conclusion： miR-494 can promote the TGF-β1 inducing secretion and deposition of extracellular matrix.
出处
《温州医学院学报》
CAS
2015年第3期162-165,170,共5页
Journal of Wenzhou Medical College
基金
国家自然科学基金资助项目(30871179/c140405)
浙江省卫生高层次创新人才培养工程项目(浙卫发[2010]190号文件)
浙江省钱江人才计划项目(2011R1083)
