摘要
目的 探讨建立人诱导性多能干细胞（iPSC）及其在体外快速高效诱导分化为肝细胞样细胞的可能.方法 用核转染的方法将含有八聚体结合转录因子3/4（Oct3/4）和p53短发卡RNA（shRNA）、转录因子性别决定区Y框蛋白（Sox）2和Kruppe样因子4（KLF4）、LMYC和LIN28的质粒导入健康人的皮肤成纤维细胞中,将其重编程为iPSC后并通过细胞碱性磷酸酶染色、细胞免疫荧光染色及反转录-聚合酶链反应（RT-PCR）技术对建立的iPSC进行鉴定.在体外,按照肝脏正常发育的生长进程,将iPSC向肝系细胞方向分化,于倒置显微镜下连续观察iPSC的形态学变化,并采用细胞免疫荧光染色方法检测各分化阶段的诱导细胞中特异性细胞分子标志表达.结果 成功获得与人胚干细胞相似形态特征的iPSC,并且碱性磷酸酶染色呈阳性,细胞免疫荧光染色及RT-PCR结果均显示建立的iPSC表达干细胞多能性标记分子Oct3/4、Sox2、Nanog、阶段特异性抗原4（SSEA4）和肿瘤排斥抗原-1-60（TRA-1-60）.在体外研究中,iPSC可成功被定向诱导分化为肝细胞样细胞,且70％ - 80％的诱导细胞表达肝细胞特异性标记蛋白白蛋白（ALB）及唾液酸糖蛋白受体1（ASGPR1）.结论 iPSC能够在体外高效分化为有功能的肝细胞样细胞.
Objective To explore the establishment of human induced pluripotent stem cells and the possibility of high efficient differentiation into hepatocyte-like cells from induced pluripotent stem cells （iPSCs） in vitro.Methods Human iPSCs were generated from healthy human dermal fibroblasts by nucleofection with the three expression plasmids encoding octamer transcription factor 3/4 （Oct3/4） and p53 short hairpin RNA （shRNA）,sex determining region Y-box 2 （Sox2） and Kruppel-like factor 4 （KLF4）,LMYC and LIN28,then the iPSCs were identified by alkaline phosphatase staining,immunofluorescence staining and reverse transcriptase-polymerase chain reaction （RT-PCR）.In vivo,the iPSCs were introduced to differentiate to hepatocytes according to the development of normal liver,and the morphological changes of induced cells were observed under the inverted microscope continually,and the specific molecular markers expression was detected in various stages from stem cells to the induced hepatocyte-like cells by immunocytochemistry staining.Results The iPSCs line had been established from human fibroblasts and the morphology was similar to that of human embryonic stem cells.The iPSCs were positive in alkaline phosphatase staining and could express stem cell pluripotent markers Oct3/4,Sox2,Nanog,stage-specific embryonic antigen 4 （SSEA4） and tumor rejection antigens-1-60 （TRA-1-60） by immunofluorescence staining and RT-PCR.In thein vitro study,the iPSC could be high efficiently differentiated into hepatocyte-like cells,and 70%-80% of the cells could express proteins that were characteristic of hepatocytes including albumin （ALB） and asialoglycoprotein receptor 1 （ASGPR1）.Conclusion The iPSCs can differentiate high efficiently into functional hepatocyte-like cells in vitro.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第10期2306-2308,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金（81370581）
湖北省自然科学基金（2015CFB573）
关键词
诱导性多能干细胞
胚干细胞
肝细胞样细胞
Induced pluripotent stem cell
Embryonic stem cells
Hepatocyte-like cells
