毛状分裂增强子1诱导肝干细胞向胆管细胞的分化  

Increased Hes1 expression to regulate differentiation of liver epithelial progenitor cells into cholangiocytes

作　　者：张竞文[1] 于兵 徐方[1] 李亚宁 胡以平 ZHANG Jingwen;YU Bing;XU Fang;LI Yaning;HU Yiping(School of Basic Medical Science,NHC Key Laboratory of Metabolic Cardiovascular Diseases Research,Ningxia Medical University,Yinchuan 750004;Department of Cell Biology,Naval Medical University,Shanghai 200003,China)

机构地区：[1]宁夏医科大学基础医学院,代谢性心血管疾病研究重点实验室,宁夏银川750004 [2]海军军医大学细胞生物学教研室,上海200003

出　　处：《西安交通大学学报（医学版）》2023年第4期505-510,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：宁夏回族自治区重点研发一般项目(No.2019BEG03006);宁夏回族自治区自然科学基金项目(No.2022AAC03192)。

摘　　要：目的探讨毛状分裂增强子1(hairy enhancer of split 1,Hes1)调控肝干细胞(liver epithelial progenitor cells,LEPCs)向胆管细胞分化的作用。方法将pTRE2hyg-Hes1和pTRE2hyg-EGFP-Hes1转染LEPCs,不同浓度强力霉素(doxycycline,DOX)诱导Hes1的表达。通过Western blotting、PCR、RT-PCR、Real-time PCR、免疫细胞化学和免疫荧光技术检测相关分子表达。以谷胱甘肽合成酶(glutathione synthetase,Gss)作为肝细胞分化的标记,以角蛋白19(keratin 19,Krt19)和肝核因子1β(hepatic nuclear factor 1β,HNF1β)为胆管细胞分化标记。结果RT-PCR和Real-time PCR检测结果显示,随着DOX浓度的增加,Hes1在LEPCs细胞中的表达逐渐增高,当DOX浓度为10μg/mL时,Hes1表达是未加DOX时的11.21倍,第5天Hes1的表达处于高峰,持续到第7天;Western blotting检测结果显示HES1蛋白表达也随着DOX浓度的增高而有所增加,HES1表达明显高于未经处理的LEPCs(P<0.05);免疫细胞化学染色显示,转染了pTRE2hyg-Hes1的细胞98%以上呈阳性染色,见于细胞核和细胞质。当Hes1在LEPCs细胞中表达上调后,Gss表达有所下降,Krt19和HNF1β表达增加。结论在pTet-on和pTRE2hyg-Hes1双转染的LEPCs中,Hes1上调可诱导LEPCs分化为胆管细胞。Objective To use hairy enhancer of split 1(Hes1)to regulate the differentiation of liver epithelial progenitor cells(LEPCs)into cholangiocytes.Methods The vectors,pTet-on and pTRE2hyg-Hes1,were transfected into LEPCs.The expression of Hes1 was induced by doxycycline(DOX)with different concentrations(0,0.1,1,5,10,50,100 and 500μg/mL).The expressions of Hes1,molecular markers of hepatocyte and cholangiocyte,glutathione synthetase(Gss),keratin 19(Krt19)and hepatic nuclear factor 1β(HNF1β)in LEPCs were verified by Western blotting,RT-PCR,Real-time PCR,immunocytochemistry and immunofluorescence.Results The expression of Hes1 in LEPCs transfected by pTet-on/pTRE2hyg-Hes1 was increased by 11.21 fold when induced by DOX at 10 ug/mL,which drove the LEPCs to differentiate into biliary epithelial cells.With increasing expression of Hes1,cholangiocyte markers,Krt19 and HNF1β,were significantly upregulated,while the hepatocyte marker,Gss,was obviously downregulated.Conclusion DOX at 10μg/mL may induce a suitably up-regulated expression of Hes1 in LEPCs double-transfected by pTet-on and pTRE2hyg-Hes1,and the suitable high-expression rather than over-expression of Hes1 can regulate LEPCs to differentiate into cholangiocytes.

关 键 词：毛状分裂增强子1 肝干细胞 分化 胆管细胞 角蛋白19 谷胱甘肽合成酶 肝核因子1β 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]