胚胎干细胞直接向神经细胞诱导分化的实验研究  

Direct differentiation of embryonic stem cells into neural cells without embryonic body culture period in vitro

作　　者：肖迎[1] 唐仕波[1] 黄冰[1] 王琪[1] 孟晶[1] 林少芬[1] 

机构地区：[1]中山大学中山眼科中心,广东广州510060

出　　处：《中国病理生理杂志》2006年第11期2081-2085,共5页Chinese Journal of Pathophysiology

基　　金：国家杰出青年基金资助项目(No.30125041);教育部博士点专项科研基金资助项目(No.A032003149);广东省教育厅"千百十工程优秀人才"培养基金资助项目(No.Q02027);中国博士后科学基金资助项目(No.2004036520)

摘　　要：目的:探讨不经拟胚体(EB)培养阶段,应用维甲酸(RA)在体外直接将胚胎干细胞(ESC)诱导分化为神经细胞的可行性和效果。方法:ESC消化后分为A、B、C、D 4组,A、B组进行EB培养后,A组用RA进行诱导分化,B组不添加RA使其自由分化,C、D组不经EB培养阶段,C组直接用RA诱导分化,D组自由分化。倒置相差显微镜及扫描电镜观察细胞形态变化,免疫细胞化学及流式细胞术观察各组第9 d分化细胞微管相关蛋白-2(MAP-2)和胶质纤维酸性蛋白(GFAP)的表达情况。结果:A、C组诱导分化的神经样细胞逐渐增多,并形成密集的神经网络样结构,9 d时大部分为MAP-2阳性细胞,MAP-2阳性率显著高于B、D组(P<0.01)。B、D组以圆形上皮样分化细胞为主,9 d时绝大部分为GFAP阳性细胞,GFAP阳性率显著高于A、C组(P<0.01)。A、C组之间或B、D组之间的MAP-2及GFAP阳性率均无显著差异(P>0.05)。结论:在体外,可不经EB阶段,应用RA直接将ESC诱导分化为神经细胞,改良的ESC诱导分化方法与传统的RA4-/4+方法效果相同,可取代传统方法。AIM： To investigate the feasibility and effect of directly differentiation of embryonic stem cells （ESC） into neural cells induced by retinoid acid （RA） without embryonic body （EB） culture period in vitro. METHODS： ESC were digested and divided into 4 groups： group A and B were undergone EB culturing. After that, cells in group A were induced by RA, cells in group B were differentiated spontaneously, cells in group C were committedly induced by RA directly without EB culturing, and cells in group D were differentiated spontaneously without EB period. Morphologic changes were observed under inverted microscope and scanning electron microscope. MAP -2 and GFAP were detected by immunocytochemistry and flow cytometry after differentiated for 9 days. RESULTS ： In groups A or C, neuronlike cells increased gradually, forming neural network. At the 9th day, a large part of cells in these groups were MAP -2 positive cells, and the positive rate was higher than that in groups B or D （P 〈0. 01 ）. Groups B and D were almost epithelial - like cells. At the 9th day, GFAP positive cells were predominant. The rate of GFAP staining cells in groups A or C was significantly lower than that in groups B or D （ P 〈 0. 01 ）. There was no significant difference of MAP - 2 or GFAP positive rates between group A and C, nor between group B and D （ P 〉 0. 05）. CONCLUSION ： ESC was directly induced into neural cells by RA without EB culture period in vitro. This modified method has the same effect as the traditional RA 4 -/4 ＋ assay and can replace the traditional method.

关 键 词：干细胞 细胞分化 细胞培养 神经元 

分 类 号：R363[医药卫生—病理学]