胚胎干细胞直接向神经细胞诱导分化的实验研究

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。

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

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,neuron-like 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.