摘要
背景与目的:采用体外获得性表达外源发状分裂相关增强子-1(hairy and enhancer of split1,Hes1)基因的方法,探讨Hes1在肝干细胞分化以及胆管上皮细胞发育中的作用。材料与方法:通过PCR方法从小鼠基因组中克隆Hes1基因片段,构建表达载体pEGFP-C1-Hes1和pcDNA3.1-Hes1,将2种表达载体分别转染肝原始细胞系(LEPCs),应用RT_PCR和Real-timePCR技术检测胆管细胞分子标志物CK19、GGT,胆管上皮细胞相关转录因子HNF6、HNF1β,肝细胞分子标志物GS、BGP和肝卵圆细胞的分子标志物Thy-1的表达,并在荧光显微镜下观察EGFP标记的LEPCs细胞系荧光强度的变化。结果:成功构建表达载体pEGFP-C1-Hes1和pcDNA3.1-Hes1。RT-PCR和Real_timePCR检测均表明胆管细胞分子标志CK19、GGT表达量上调;胆管上皮细胞相关转录因子HNF6、HNF1β表达上调;肝细胞分子标志GS、BGP表达量下调;卵圆细胞的分子标志Thy-1表达量下调;LEPCs细胞系绿色荧光增强。结论:初步证明小鼠肝原始细胞经Hes1的表达诱导后可向胆管上皮细胞方向分化,推测Hes1为胆管上皮细胞分化的转录调控因子。
BACKGROUND AND AIM： To study the differemiation of mouse liver epithelial progenitor cells（LEPCs） to cholangiocytes induced by hairy and enhancer of split 1（Hes1）gene in vitro. MATERIALS AND METHODS： The Hesl gene was amplified by PCR from mouse tail genomic DNA and subcloned into pEGFP-C1 and pcDNA3.1 vector. LEPCs were transfected and performed in vitro. Cholangiocyte markers, CKIg, GGT, HNF6 and HNF1β; hepatocyte markers, GS and BGP; hepatic oval cell marker, Thy-1 were assayed with RT-PCR and Real-time PCR. The fluorescence of the LEPCs tagged with EGFP was observed in vitro. RESULTS： Cholangiocyte markers, CK19, GGT, HNF6 and HNF1β, were up-regulated. Hepatocyte markers GS and BGP, and hepatic oval cell marker Thy-1, were down- regulated. CONCLUSION： It indicated that LEPCs differentiated to cholangiocytes initially in vitro, and Hesl might play important roles in the differentiation of LEPCs and influence cholangiocyte versus hepatocyte fate determination.
出处
《癌变．畸变．突变》
CAS
CSCD
2008年第6期424-428,共5页
Carcinogenesis,Teratogenesis & Mutagenesis
基金
国家自然科学基金项目(30470876,30600326,30472141)
国家863计划项目(2006AA02Z474)
上海重点基础科学计划研究项目(03DJ14020,06DJ14001)
关键词
HES1
肝原始细胞
分化
胆管上皮细胞
Hes1
liver epithelial progenitor cell
differentiation
cholangiocyte
