CAJ | 학술논문

目的建立白蛋白(Alb)和角蛋白19 (CK19)双标记的胚胎干细胞系,并探讨其在肝祖细胞分化中的应用. 方法构建含有Alb启动子的绿色荧光蛋白载体,并带有新霉素抗性,同时构建由CK19启动子启动红色荧光的载体,带有潮霉素抗性；将构建好的载体线性化后,同时电转胚胎干细胞El4.1,利用新霉素和潮霉素双筛选后,建立双标记基因修饰胚胎干细胞系El4.1-2；通过拟胚体形成及生长因子(骨形态发生蛋白4和碱性成纤维细胞生长因子)刺激的方法诱导产生肝祖细胞,经过流式细胞术和逆转录-聚合酶链反应检测诱导效率.结果构建了含有Alb启动子启动绿色荧光和CK19启动子启动红色荧光的载体,并证明了Alb和CK19启动子能特异启动各自蛋白的表达；建立了Alb和CK19双标记胚胎细胞系,并证明其具有多能性,显著表达多能性基因八聚体结合转录因子4和阶段特异性胚胎抗原1,在El4.1-2诱导分化22d后通过流式细胞术分选成功得到21.27％ Alb和CK19双阳性的肝祖细胞.结论双标记胚胎干细胞的建立可以纯化和定量肝祖细胞,便于改进肝向诱导的方法及研究肝祖细胞特性.
목적 건립백단백(Alb)화각단백19 (CK19)쌍표기적배태간세포계,병탐토기재간조세포분화중적응용. 방법 구건함유Alb계동자적록색형광단백재체,병대유신매소항성,동시구건유CK19계동자계동홍색형광적재체,대유조매소항성；장구건호적재체선성화후,동시전전배태간세포El4.1,이용신매소화조매소쌍사선후,건립쌍표기기인수식배태간세포계El4.1-2；통과의배체형성급생장인자(골형태발생단백4화감성성섬유세포생장인자)자격적방법유도산생간조세포,경과류식세포술화역전록-취합매련반응검측유도효솔.결과 구건료함유Alb계동자계동록색형광화CK19계동자계동홍색형광적재체,병증명료Alb화CK19계동자능특이계동각자단백적표체；건립료Alb화CK19쌍표기배태세포계,병증명기구유다능성,현저표체다능성기인팔취체결합전록인자4화계단특이성배태항원1,재El4.1-2유도분화22d후통과류식세포술분선성공득도21.27％ Alb화CK19쌍양성적간조세포.결론 쌍표기배태간세포적건립가이순화화정량간조세포,편우개진간향유도적방법급연구간조세포특성.
Objective To establish a gene-modified embryonic stem (ES; E14.1-2) cell line with hepatoblast differentiation reporter genes,albumin (ALB) and cytokeratin 19 (CK19),labeled to facilitate study of their potential applicability as differentiated hepatoblasts.Methods Two expression vectors were constructed,one with the ALB promotor driving the enhanced green fluorescent protein (EGFP) and antineomycin genes (pA1b-EGFP),and the other with the CK19 promotor driving the red fluorescence protein and anti-hygromycin genes (pCK19-hCD25-IRES-tdTOMATO).The linearized vectors were electroporated into the E14.1 line,and double reporter genes-modified ES cells (E14.1-2) were selected by neomycin and hygromycin.E14.1-2 hepatoblast differentiation was indcued by exposure to growth factors (BMP4 and bFGF) and evidenced by embryoid body formation.Fluorescence-activated cell sorting (FACS) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm whether differentiated cells were hepatoblast-like and to quantify the differentiation efficiency.Results The pAlb-EGFP and pCK19-hCD25-IRES-tdTOMATO vectors were shown to specifically activate ALB and CK19 exprssion.The E14.1-2 cell line with labeled ALB and CK19 was established,and shown to have pluripotency by RT-PCR detection of pluripotent markers' expression,namely Oct4 and SSEA-1.After 22 days of induction,21.27％ of the differentiated hepatoblasts were detected by FACS as positive for ALB and CK19 expression.Condusions A gene-modified ES cell line was generated with hepatocyte differentiation reporter genes ALB and CK19 labeled.The differentiation of the resultant E14.1-2 line was technically simple to qualify and quantify,and will likely aid future studies of hepatoblast characteristics.