人胚胎干细胞神经分化过程中VEGF作用机制的研究

目的观察血管内皮生长因子(vascular endothelial growth factor,VEGF)对人胚胎干细胞分化为神经元的促进作用是否与Notch信号通路有关。方法人胚胎干细胞经拟胚体向神经元分化,并分为3组:A组为常规诱导组;B组为常规诱导+VEGF(10ng/ml)作用组;C组为常规诱导+γ-分泌酶抑制剂预处理+VEGF(10ng/ml)作用组。用免疫荧光法检测及计算各组不同阶段细胞阳性率,半定量RT-PCR观察各组神经干细胞Hes1 mRNA表达,MTT法检测3组神经干细胞增殖速率。结果免疫荧光法检测显示,B组产生神经干细胞的阳性率明显高于A、C两组,差异有统计学意义(P0.01),A、C两组之间无显著性差异(P0.05);B、C两组进一步分化为神经元的阳性率均明显高于A组,差异有统计学意义(P0.01),B、C两组之间无显著性差异(P0.05);半定量RT-PCR观察显示B组神经干细胞Hes1 mRNA表达明显高于A、C两组;MTT法检测与半定量RT-PCR结果相似。结论人胚胎干细胞体外分化过程中血管内皮生长因子通过激活Notch信号通路,促进神经干细胞增殖,而VEGF在神经干细胞向神经元分化中的作用与Notch通路无关。

The Mechanism of VEGF on Neural Differentiation of Human Embryonic Stem in vitro

Objective To study the mechanism of VEGF on the neural differentiation of hESCs in vitro.Methods hESCs were cultured and hESCs differentiated into neurons through the embryonic body.Before the VEGF（10ng/mL） was added,γ-secretase inhibitor was given at every stage of induction for 1h.The markers of every stage were detected and the percentages of the imunostaining positive cells were counted through immunofluorescence.The expression of Hes1 mRNA in each group was detected by RT-PCR.Results The percentage of Nestin in the γ-secretase inhibitor pretreatment group was similar with the conventional group.RT-PCR revealed that the expression of Hes1mRNA in the VEGF-treated group was higher than those in the γ-secretase inhibitor preconditioning group and the conventional group.At the differentiation of NSCs into mature neurons,the neurons generated from γ-secretase inhibitors group and the VEGF-treated group had no significant difference.Conclusions Through the activation of Notch signal transduction pathway,VEGF play a role in promoting human embryonic stem cell differentiating into neural stem cells.The inhibitor of Notch signaling can antagonize the effect of VEGF in this process.