人胚胎干细胞神经分化的方法探讨  

Neural Differentiation of Human Embryonic Stem Cells

作　　者：赖文玉[1] 曾巧慧[1] 吴燕云[1] 成福义[2] 李攀龙[2] 李伟强[2] 

机构地区：[1]中山大学附属第二医院儿科,广东广州510120 [2]中山大学中山医学院,广东广州510080

出　　处：《中山大学学报（医学科学版）》2011年第5期694-700,共7页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金博士启动项目(9451008901002230)

摘　　要：【目的】利用新建系的人胚胎干细胞株SYSU-7,运用胚体形成的方法,探讨不同的分化条件对胚胎干细胞神经分化的影响。【方法】首先检测人胚胎干细胞株SYSU-7的Oct4、SSEA4(stage-specific embryonic antigen-4)、TRA-1-60和AKP(Alkaline phosphatase)等胚胎干细胞特异性标志物的表达及体内外三胚层分化的能力;之后,应用含20%KnockoutSerum Replacement(KSR)的培养液和神经干细胞培养液(neural progenitor medium,NPM)分别诱导细胞形成悬浮的拟胚体(悬浮时间为4 d或7 d),然后将拟胚体贴壁于多聚鸟氨酸(Polyornithine,PO)和纤维粘连蛋白(Fibronectin,FN)包被的培养皿中继续培养4～10 d。在分化的不同时间点利用免疫荧光染色的方法检测胚胎干细胞和神经细胞相关的标志性抗原。【结果】SYSU-7胚胎干细胞表达未分化细胞特异性标志Oct4、SSEA4,TRA-1-60,AKP染色呈强阳性,在体内外具有向三胚层分化的潜能;在体外诱导其向神经分化,结果发现KSR培养液比神经干细胞培养液更有利于胚胎干细胞的神经分化,悬浮生长7 d的拟胚体贴壁后出现特征性的玫瑰花环样结构(rosette structure)的时间更早、数量更多。【结论】SYSU-7细胞系具有自我更新及多向分化潜能等胚胎干细胞特性。本实验为研究人胚胎干细胞的神经分化提供了新的细胞模型和研究思路。【Objective】 To explore the neural differentiation potential of a new human embryonic stem cell line,SYSU-7,through embryoid body formation under different culture condition.【Methods】 Detail characterization of SYSU-7 cells were carried out by detecting the expression of Oct4,SSEA4,TRA-1-60,AKP,and multi-lineage differentiation capacity in vivo and in vitro.20% KSR medium（KSR） and neural progenitor medium（NPM） were used to induce neural differentiation of SYSU-7 cells,respectively.Embryoid bodies（EBs） were generated and cultured in suspension for 4d or 7d before attached to polyornithine and fibronectin（PO＋FN） coated culture dishes.Neural stem/progenitor cells derived from SYSU-7 cells were identified by the immunocytochemical assays.【Results】 The immunostaining analysis showed that SYSU-7 cells expressed the undifferentiated marker Oct4,SSEA4,and TRA-1-60,as well as the AKP.The results also demonstrated that SYSU-7 cells have the capacity to differentiate into three germ layers both in vitro and in vivo.KSR medium was found to be superior to NPM for neural differentiation of SYSU-7 cells.EBs cultured in suspension for 7 d resulted in earlier appearance and greater amount of rosette structures than 4-day EBs.【Conclusion】 SYSU-7 cells possessed the distinct characteristics of human embryonic stem cells,self-renew and pluripotency.The results provided a new cell model and may have implications for neural differentiation studies.

关 键 词：人胚胎干细胞 神经分化 

分 类 号：Q254[生物学—细胞生物学]