胶质细胞源性神经营养因子诱导神经干细胞分化过程中bHLH转录因子的表达变化

Expresstion change of bHLH transcription factors during neural stem cells differentiation induced by glial cell line-derived neurotrophic factor

目的观察胶质细胞源性神经营养因子（glial cell line-derived neurotrophic factor，GDNF）对神经干细胞分化的影响，探讨碱性螺旋环螺旋（basic helix-loop-helix，bHLH）转录因子是否参与在此过程中。方法无菌条件下取孕14。16d胎鼠端脑进行神经干细胞体外培养，将第三代神经球分为对照组和GDNF组，分化1、3、7d细胞进行免疫荧光染色并计算β-tubulinIII阳性率，荧光定量PCR技术检测bHLH基因Hes-1、Hes．5、Mash-1的表达变化。结果免疫荧光染色结果显示，分化1、3、7d时，β-tubulinlll阳性细胞比率分别为：对照组：（3．76±1．14）％、（7．40±1．25）％、（12．65±1．58）％；GDNF组：（7．29±1．57）％、（19．80±1．67）％、（27．55±2．03）％，GDNF组神经元分化率明显高于对照组（P〈0．05）。荧光定量PCR结果显示，Hes-1和Hes-5在分化1、3、7d均保持极低表达，各时间点间比较无明显差异；Mash-1表达则在分化1、3、7d内持续上升，各时间点间比较差异有统计学意义（P〈0．05）。结论GDNF能通过Mash-1的激活来诱导神经干细胞分化为更多的神经元，并且此过程中bHLH转录因子表达有明显特异性，将为日后进行神经干细胞定向分化的机制研究奠定基础。

Objective Observe the effect of glial cell line-derived neurotrophic factor （GDNF） the differentiation of neural stem cells, to explore if the basic helix loop helix （bHLH） transcription factors are involved in this process or not. Methods Separated the telencephalon of rat embryos in sterile condition and cultured in vitro, pick up the third generation neurosphere and divided into control group and GDNF group to induced differentiation. Immunofluorescence staining calculated the cells which had differentiated 1,3,7 days, and RQ-PCR detected, the expression changes of genes Hes-1, Hes-5 and Mash-1 of these cells. Results The mmunofluorescence staining suggested that after differentiated 1,3,7 days the proportions in control group β-tubulin III positive cell were （3.76±1.14）%, （7.40±1.25）%, （12.65±1.58）%; the GDNF group were （7.29±1.57）%, （19.80±1.67）%, （25.55± 2.03 ）%, Neuronal differentiation of GDNF group was significantly higher than the control group （P〈0.05）. RQ-PCR suggested that Hes-1 and Hes-5 kept a very low expression in 1,3,7 day after differentiation, there were no obvious difference between each time point.Mash-1 expression kept rising in 1,3,7 day after differentiation, there were statistically significant difference between each time point （P〈0.05）. Conclusion GDNF can activate bHLH transcription factor induced neural stem cells into β-tubulin III posi- tive cell differentiation, and in this process, the expression of the bHLH transcription factor has obvious specificity, thses results would lay a foundation for the research of mechanism of differentiation of neural stem cells.