目的 观察黄芪总皂甙(TSA)调控体外培养大鼠神经干细胞(NSCs)向神经元方向分化的作用及其有效浓度,并研究其Wnt/β-catenin信号通路相关机制。方法 新生24 h大鼠大脑皮层来源NSCs进行原代、传代培养并鉴定,通过CCK-8试剂盒初筛TSA诱导分化的可能有效浓度。取第三代NSCs,设不加TSA的正常组和不同浓度TSA实验组,分化培养7 d后,间接免疫荧光法和Western blotting检测微管相关蛋白2 (MAP-2)和胶质纤维酸性蛋白(GFAP)表达。另设正常组、最佳浓度TSA组、TSA+Wnt/β-catenin信号通路抑制剂ICG-001组和ICG-001组,分化培养7 d后,Western blotting检测各组Wnt3/3a、β-catenin和抗神经原素1 (Ngn1)蛋白表达。结果 TSA在浓度1×10-4 mol/L、1×10-5 mol/L、1×10-6 mol/L时,NSCs数量显著增加(P < 0.001)。TSA能增加NSCs分化为神经元的比例,1×10-5 mol/L最佳(P < 0.05)。TSA能上调Wnt3/3a、β-catenin与Ngn1蛋白的表达(P < 0.05)。结论 TSA能促进体外培养NSCs向神经元方向分化,与激活Wnt/β-catenin信号通路,从而调控该通路下游促分化靶向蛋白Ngn1的表达有关。TSA最佳促神经元方向分化浓度约为1×10-5 mol/L。
Objective To observe the effects of total saponins of Astragalus (TSA) on neural stem cells (NSCs) differentiating to neuron in vitro and the best concentration, as well as the role of Wnt/β-catenin signaling pathway in it. Methods NSCs from the cerebral cortex of a neonatal rat were cultured and identified. The possible effective concentration of TSA was screened with Cell Counting Kit-8 (CCK-8). The third generation of NSCs was induced as normal group (without TSA) and various concentration of TSA groups for seven days. The expression of microtubule-associated proteins (MAP-2) and glial fibrillary acidic protein (GFAP) was detected with indirect immunofluorescence and Western blotting to detect the ratio of neuron and astrocyte. Then, the third generation of NSCs was induced as normal group (without TSA), the best concentration of TSA group, TSA group and inhibitor ICG-001 group, and ICG-001 group, for seven days, and the expression of Wnt3/3a, β-catenin and neurogenin 1 (Ngn1) was detected with Western blotting. Results TSA promoted the NSCs proliferation in the concentration of 1×10-4 mol/L, 1×10-5 mol/L and 1×10-6 mol/L (P < 0.001), and increased the proportion of neurons, especially in the concentration of 1×10-5 mol/L (P < 0.05). TSA increased the expression of Wnt3/3a, β-catenin and Ngn1. Conclusion TSA could promote NSCs differentiating into neurons in vitro, which may associate with the activation of the Wnt/β-catenin signaling pathway and the expression of differentiation-promoting target protein Ngn1. TSA can do it best in the concentration about 1×10-5 mol/L..
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