Hes1基因对人γ-氨基丁酸能神经元分化的影响

The effect of hairy and enhancer of split 1 on human gamma aminobutyric acid neurons’ differentiation

目的观察单因素抑制Hes1基因的表达对人神经干细胞诱导分化为γ-氨基丁酸（GABA）能神经元分化率的影响。 方法对人神经干细胞进行原代培养，传代（神经干细胞5～7 d传代1次）。取传至第2代的神经干细胞进行神经元巢蛋白免疫荧光鉴定，证明其为神经干细胞后进行GABA能神经元的诱导分化。采用完全随机分组原则将第二代神经干细胞分为对照组和实验组。对照组为含胎牛血清的普通诱导分化液，实验组除加入抑制Hes1基因的寡核苷酸链外，其余成分同对照组相同。待诱导分化结束后分别对实验组及对照组进行GABA能神经元免疫荧光染色鉴定。 结果诱导分化后第6天，约半数以上细胞开始分化并出现汇聚现象，重新消化接种，第14天镜下可见90%～95%细胞已贴壁完成分化。进行GABA能神经元免疫荧光染色鉴定，随机选取10个视野，实验组每个视野下在100个细胞中可见55～70个GABA阳性细胞，分化率可达（64.0±0.6）%，而对照组在同样情况下仅可见15～20个GABA阳性细胞，分化率仅为（16.6±0.6）%，两者比较差异有统计学意义。 结论抑制Hes1基因的表达可以显著提高人神经干细胞诱导分化为GABA能神经元的分化率。

ObjectiveTo investigate the effect of blocking hairy and enhancer of split 1 （Hes1） on gamma aminobutyric acid （GABA） neurons’ differentiation in human neural stem cells. MethodsThe human neural stem cells were cultured for 10 days and identified by nestin immunofluorescence stain, then using the human neural stem cells to differentiate GABA neurons. The design project of experiment was adopted completely random. The control cultured medium were the common differentiated medium added 10% bovine serum albumin. And the experimental cultured mediums were identical with the control excepted for the anti-Hes1. Anti-GABA immunofluorescence stain was carried out when most of the cells were differentiated. ResultsA lot of 150-200 μm diameter neurosphere which were identified when cultured for 10 days. The second generation of neurospheres were using for GABA neurons differentiation. At the sixth day, more than half of cells were differentiated and begin to concentrate. Digested and inoculated again, then 90%-95% cells were adherent and differentiated at day 14. Anti-GABA immuno-fluorescence stain showed that 55-70 positive cells were be found in 100 cells in every visual field, the rate was （64.0±0.6）% in anti-Hes1 differentiated group and in the same condition the positive cells were only 15-20 positive cells, the rat was （16.6±0.6）% in control group, there are significant difference between the two groups. Conclusionblocking Hes1 can significant increase the differentiation rate of GABA neurons in human neural stem cells.