改进传代诱导法高效诱导人胚胎干细胞定向分化为内胚层细胞  

Improved passage induction method efficiently induces human embryonic stem cells to differentiate into endoderm cells

作　　者：崔罗方 王亚娜 宋晓聪[1] 闫萌萌 马文华[1] 许晓琳[1] 尤雪剑[1] 刘丽[1] 张艳[1] 安思琪 CUI Luo-fang;WANG Ya-na;SONG Xiao-cong;YAN Meng-meng;MA Wen-hua;XU Xiao-lin;YOU Xue-jian;LIU Li;ZHANG Yan;AN Si-qi(Cangzhou Medical School,Cangzhou 061000)

机构地区：[1]沧州医学高等专科学校,沧州061000

出　　处：《生殖医学杂志》2021年第2期230-235,共6页Journal of Reproductive Medicine

基　　金：沧州市级课题(183302109)。

摘　　要：目的比较不同诱导方法将人类胚胎干细胞(hESCs)诱导分化为纯度较高的人类内胚层细胞的分化效率,探寻最佳的分化体系。方法用两种不同诱导方法培养人类胚胎干细胞(hESCs)H9细胞株,第1组采用最新改进的体外传代诱导方法,用IV型胶原酶消化成团的hESCs为单个hESC细胞后,使用含有100 ng/ml的胚胎干细胞诱导因子活化素A(activin A)的分化诱导液体外诱导hESCs 3 d(改良组);第2组采用传统方法,直接在未经消化hESCs培养液中添加诱导因子activin A(传统组)。分化完毕后免疫荧光检测两组细胞中内胚层标志物SOX17和叉头框A2(FOXA2)表达水平。结果改良组的分化细胞具有类似内胚层细胞的形态结构,细胞体积变大,细胞之间界限清晰,形态呈现多角形,而传统组的细胞呈团状聚集生长,彼此紧密排列,细胞与细胞之间界限不清楚,细胞形态没有明显的变化。细胞免疫荧光化学检测结果显示改良组的细胞中内胚层标志物SOX17和FOXA2的表达率较传统组显著升高(P<0.05)。结论采用最新改进的体外传代诱导法,activin A可诱导hESCs分化为高纯度的内胚层细胞,与传统方法相比较,此种新方法诱导内胚层细胞的效率明显升高。Objective:To compare the differentiation efficiency of human embryonic stem cells(hESCs)induced by different induction methods into human endoderm cells with high purity,and to explore the best differentiation system.Methods:Two different induction methods were used to culture the H9 cell line of human embryonic stem cells(hESCs).In the first group,an improved induction method was used.hESCs were digested with type IV collagenase to single hESC cell.hESCs were induced by culture medium containing 100 ng/ml activin A for 3 days to induce hESCs to endoderm cells.In the second group,traditional induction method was used.Activin A was added into undifferentiated hESCs.Two groups of cells were collected after differentiation.Cell immunofluorescence was used to detect the endodermal marker SOX17(SRY—related HMG-box gene 17)and FOXA2(Forkhead box transcription factor A2).Results:At the end of the induction,the differentiated cells in the improved induction method group showed an endoderm-like morphology.The cell volume increased,the boundaries between cells were clear,and the morphology of the cells was polygonal.In the traditional induction method group,the cells were clustered,the boundaries between cells were not clear,and there was no obvious change in cell morphology.The results of immunofluorescence showed that expression of SOX17 and Foxa2 in the improved induction method group was significantly higher than the traditional induction method group(P<0.05).Conclusions:Using the latest improved in vitro passage induction method,activin A can induce hESCs to differentiate into high-purity endoderm cells.Compared with traditional methods,this new method has a significantly higher efficiency in inducing endoderm cells.

关 键 词：胚胎干细胞 内胚层细胞 诱导 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]