血根碱抑制TGF-β1诱导人脐静脉内皮细胞向间质细胞转化研究  

Inhibitory effect of Sanguinarine on TGF-β1-induced endothelial-mesenchymal transition of HUVEC

作　　者：胡哲夫[1] 唐其柱[1] 刘源[1] 李金[1] 张文斌[1] 袁园[1] 

机构地区：[1]武汉大学附属人民医院心内科

出　　处：《中华生物医学工程杂志》2015年第4期314-318,共5页Chinese Journal of Biomedical Engineering

基　　金：教育部博士点基金（20130141130010）

摘　　要：目的：研究血根碱（SAN）对TGF-β1诱导人脐静脉内皮细胞（HUVEC）向间质细胞转化的影响，并探讨其发生机制。方法使用TGF-β1（10μg/L）刺激HUVEC，观察不同浓度SAN对TGF-β1诱导HUVEC向间质细胞转化的影响。采用CCK8检测不同浓度SAN对HUVEC细胞活性的影响；在倒置相差显微镜下观察HUVEC形态学的改变；采用实时半定量逆转录-聚合酶链反应（RT-PCR）检测细胞中collagen-1，fibronectin，N-cadherin的基因表达水平；免疫印迹法检测细胞中p-Smad2，Smad2，p-Smad3，Smad3的蛋白表达量。结果 CCK8结果显示：不同浓度SAN干预HUVEC 72 h后，细胞活性无明显改变；光学显微镜下观察到HUVEC在正常情况下呈规则的铺路石样生长，TGF-β1作用于细胞后可见细胞形态向长梭形转变，而当SAN（0.25μmol/L）与TGF-β1同时作用时则可显著逆转这一现象；RT-PCR结果显示TGF-β1可显著上调HUVEC胞内collagen-1，fibronectin，N-cadherin的基因表达水平，当SAN与TGF-β1同时作用时，可明显逆转这种现象；WB结果显示，在TGF-β1的作用下，p-Smad2和p-Smad3表达增加，而当SAN与TGF-β1同时作用时可逆转这一现象。结论 SAN可抑制TGF-β1诱导的HUVEC向间质细胞转化，提示其对于治疗心肌纤维化疾病可能具有重要的作用。Objective To investigate the effect of Sanguinarine （SAN） on TGF-β1-induced endothelial-mesenchymal transition （EMT） of human umbilical vein endothelial cells （HUVECs） and its underlying mechanism. Methods TGF-β1（10 ng/mL）was used to stimulate HUVECs to observe the effect of SAN at various concentrations on TGF-β1-induced EMT of HUVEC. CCK8 assay was employed to detect the cell viability of HUVECs stimulated by SAN. The morphology of HUVEC was observed under inverted phase-contrast microscope. The expression levels of collagen-1,fibronectin,N-cadherin in HUVEC were determined by real-time semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. Western blot was conducted to detect the expressions of p-Smad2,Smad2,p-Smad3 and Smad3 in HUVECs. Results CCK8 assay showed that the cell viability of HUVEC did not change obviously under the stimulation of SAN at various concentrations for 72 h. Optical microscopy indicated that HUVEC exhibited regular‘cobblestone’morphology under normal circumstances but switched to fusiform cells after treated with TGF-β1. This phenomenon was apparently regressed under the costimulation of TGF-β1 and SAN（0.25μM）. The results of RT-PCR showed that the expression levels of collagen-1,fibronectin,N-cadherin were upregulated with the stimulation of TGF-β1. Western blot showed that the expressions of p-Smad2 and p-Smad3 increased under the stimulation of TGF-β1,but was regressed by addition of SAN. Conclusion SAN could inhibit the development of EMT of HUVEC induced by TGF-β1,suggesting its potentially important role in the treatment of myocardial fibrosis.

关 键 词：血根碱 转化生长因子Β1 人脐静脉内皮细胞 上皮向间质转化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]