Notch信号转导通路在原代肝星状细胞体外培养活化中的作用  

The role of Notch signal transduction pathway on the activation of primary hepatic stellate cells in vitro

作　　者：潘艳[1] 张启迪 房凤梅 华香[1] 王维[1] 傅承宏 秦娟 王万鹏 PAN Yan;ZHANG Qi-di;FANG Feng-Mei;HUA Xiang;WANG Wei;FU Chen-Hong;QIN Juan;WANG Wan-peng(Department of Laboratory Medicine,LianShui People＇s Hospital,Huai＇an 225400,diangsu Province,China;Department of Gastroenterology,Shanghai First People＇s Hospital,Shanghai diaotong University,Shanghai 200080,China;Department of Nephrology,Lianshui People＇s Hospital,Huai＇an 225400,diangsu Province,China)

机构地区：[1]涟水县人民医院检验科,江苏淮安223400 [2]上海交通大学医学院附属上海市第一人民医院消化科,上海200080 [3]涟水县人民医院肾脏科,江苏淮安223400

出　　处：《中国肝脏病杂志（电子版）》2018年第3期66-72,共7页Chinese Journal of Liver Diseases：Electronic Version

基　　金：国家自然科学基金青年项目(81600479;81600549);淮安市自然科学研究计划(HAB201737)

摘　　要：目的探讨Notch信号转导通路在肝星状细胞(hepatic stellate cells,HSC)体外培养活化中的作用。方法采用两步酶灌注法分离培养原代大鼠HSC并进行体外培养。激光共聚焦显微镜检测体外培养HSC第1天和第7天时形态、胞内维生素A、静止标志胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和活化标志α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的变化。采用RTPCR和Western blot检测HSC体外培养0天、3天和7天时上皮-间充质转分化(epithelial-to-mesenchymal transition,EMT)相关分子α-SMA、Ⅰ型胶原(collegenⅠ)、N-钙黏蛋白(N-cadherin)、蜗牛家族转录抑制因子1(snail family transcriptional repressor 1,Snail 1)等和Notch信号转导通路(受体Notch 1、Notch 2、Notch 3、Notch 4与靶基因Hes1、Hey1、Hey2、HeyL)的变化。使用Notch受体阻断剂DAPT干预后,检测体外培养的HSC中活化相关分子(α-SMA、CollegenⅠ、N-cadherin和Snail 1等)的改变。结果 (1)初分离的HSC经7天体外培养后逐渐向肌成纤维细胞(myofibroblasts, MFs)样转化,表现为形态变长、维生素A水平和GFAP表达降低,α-SMA表达升高。(2)RT-PCR与Western blot结果表明,HSC体外培养存在自发EMT现象,表现为α-SMA、CollegenⅠ、N-cadherin和Snai l表达上调及E-cadherin、CK7、CK19和Desmoplakin表达下调,同时Notch 2、Notch 3及Notch靶基因Hey2、HeyL表达逐渐增加。(3)使用DAPT后,可显著抑制原代HSC体外活化,表现为α-SMA、CollegenⅠ、N-cadherin和Snail 1的下调及E-cadherin、CK 7、CK 19和Desmoplakin转录或蛋白表达水平上调。结论 Notch信号转导通路的激活可能参与HSC的活化,可能是抗肝纤维化治疗的潜在靶点。Objective To investigate the role of Notch signal transduction pathway on the activation of primary hepatic stellate cells （HSC） in vitro. Methods HSC were isolated from the liver of male rats by enzymatic perfusion. Laser scanning confocal microscope （LSCM） was carried out to detect the change of cell shape, expression of vitamin A, glial fibrillary acidic protein （GFAP） and α-smooth muscle actin （α-SMA） at day 0 and day 7 in vitro. RT-PCR and Western blot were used to detecte the expression of gene associated with epithelial-to- mesenchymal transition （EMT） [α-SMA, CollegenⅠ, N-cadherin, snail family transcriptional repressor 1 （Snail 1）, et al] and Notch signaling pathway （Notch 1, Notch 2, Notch 3, Notch 4 and their target gene Hes 1, Hey1, Hey2, HeyL） in HSC at day 0, day 3 and day 7. DAPT were administrated to inhibit Notch signaling pathway of primary HSC at day 4, and the expression of gene associated with EMT （α-SMA, CollegenⅠ, N-cadherin, Snail 1, et al） were dectected by RT-PCR and Western blot at day 7. Results ①The primary isolated HSC was cultured in vitro for 7 days and then gradually transformed to myofibroblast （MFs）, which showed that the morphology became longer, the expression of vitamin A and GFAP decreased, and the expression of α -SMA increased. ②RT- PCR and Western blot showed that spontaneous EMT occurred during the culture of HSC in vitro, characterized by the up-regulation of α-SMA, CollegenⅠ, N-cadherin, Snai l and down-regulation of E-cadherin, CK7, CK19 and Desmoplakin. The expression of Notch 2, Notch 3 and their target genes （Hey2, HeyL） increased. ③DAPT could significantly inhibit the expression mRNA or protein of α-SMA, CollegenⅠ, N-cadherin and Snail 1, et al, and could increase the expression of E-cadherin, CK-7, CK-19 and Desmoplakin, which were typical ductal epithelial cell markers. Conclusions The activation of primary HSC is closely associated with the activation of the Notch signaling pathway. Notch signaling ma

关 键 词：细胞分离 肝纤维化 上皮-间充质转分化 肝星状细胞 Notch信号转导通路 

分 类 号：R575[医药卫生—消化系统]