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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(P<0.01),A、C两组之间无显著性差异(P>0.05);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(P<0.01),B、C两组之间无显著性差异(P>0.05);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。
Objective To observe whether the promoting effect of vascular endothelial growth factor (VEGF) on the differentiation of human embryonic stem cells into neurons is related to the Notch signaling pathway. Methods Human embryonic stem cells (NSCs) differentiated into neurons by embryoid bodies were divided into 3 groups: group A was a conventional induction group; group B was induced by conventional induction of VEGF (10 ng / ml); group C was induced by conventional induction of γ- Enzyme inhibitor pretreatment + VEGF (10ng / ml) effect group. Immunofluorescence method was used to detect and calculate the positive rate of cells in different stages of each group. Semi-quantitative RT-PCR was used to observe the expression of Hes1 mRNA in each group. The proliferation rate of neural stem cells was detected by MTT assay. Results Immunofluorescence showed that the positive rate of neural stem cells in group B was significantly higher than that in group A and C (P <0.01), but there was no significant difference between groups A and C (P> 0.05 ). The positive rates of further differentiation into neurons in group B and C were significantly higher than those in group A (P <0.01). There was no significant difference between groups B and C (P> 0.05). Semi-quantitative RT-PCR showed that the expression of Hes1 mRNA in neural stem cells of group B was significantly higher than that of group A and C; MTT assay was similar to that of semi-quantitative RT-PCR. Conclusion Vascular endothelial growth factor (VEGF) promotes the proliferation of neural stem cells by activating Notch signaling pathway in vitro. The role of VEGF in the differentiation of neural stem cells into neurons is not related to the Notch pathway.