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  • Title: [Differentiation of induced pluripotent stem cells into neural stem cells induced by brain-derived neurotrophic factor via Wnt/β-catenin and extracellular signal-regulated kinase/mitogen-activated protein kinases signal pathway].
    Author: Chen SQ, Huang M, Liu CL, Shen YY, Cai Q, Wang PJ.
    Journal: Zhonghua Yi Xue Za Zhi; 2017 Nov 07; 97(41):3263-3268. PubMed ID: 29141367.
    Abstract:
    Objective: To investigate the mechanism of brain-derived neurotrophic factor (BDNF) promoting induced pluripotent stem cells (iPSCs) to differentiate into neural stem cells (NSCs) via Wnt/β-catenin and extracellular signal-regulated kinase/mitogen-activated protein kinases (ERK/MAPK) signal pathways. Methods: iPSCs were cultured and identified. The iPSCs were induced to differentiate into NSCs by BDNF and retinoic acid (RA). Nestin was detected by immunofluorescence and flow cytometry after iPSCs differentiated. The technique of small interfering RNA (siRNA) was used to silence the gene expression of β-catenin and ERK, and iPSCs were divided into control group, BDNF group (adding 10 μg/L BDNF), siRNA-ERK/BDNF group (transfected with siRNA-ERK and adding 10 μg/L BDNF) and siRNA-β-catenin/BDNF group (transfected with siRNA-β-catenin and adding 10 μg/L BDNF). Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to detect the mRNA and protein expression of key elements of Wnt/β-catenin and ERK/MAPK signaling pathways, included β-catenin, ERK1/2, c-fos, c-jun, and c-myc. The least significant difference test was used when data were compared between groups. Results: The immunofluorescence showed that iPSCs expressed octamer-binding transcription factor-4 (Oct4), SRY-related HMG box protein-2 (Sox2) and Nanog genes. The flow cytometry showed that Nestin-positive cells were 78.7% for BDNF and 43.5% for RA, and it was only 7.8% for routine medium. Compared with those in the control group, the mRNA expression of β-catenin, ERK1/2, c-fos, c-jun, and c-myc in the BDNF group were upregulated significantly (t=2.80, 2.318, 2.255, 1.799, 1.582, 1.663, all P<0.05), and the same results were acquired with the protein expression (t=2.805, 2.318, 2.255, 1.799, 1.582, 1.663, all P<0.050). Compared with those in BDNF group, the mRNA and protein expression of ERK1/2 in siRNA-ERK/BDNF group down-regulated obviously (t=1.917, 2.042, 1.673, 1.540, all P<0.05), and the mRNA and protein expression of c-fos and c-jun were down-regulated (t=1.022, 0.907, 0.848, 0.801, all P<0.05). However, the mRNA and protein expression of β-catenin and c-myc were not suppressed by siRNA-ERK (t=0.216, 0.185, 0.097, 0.112, all P>0.05). In siRNA-β-catenin/BDNF group, the mRNA and protein expression of β-catenin and c-myc was obviously down-regulated when compared with those in BDNF group (t=3.104, 2.774, 2.235, 1.911, all P<0.05), and expression of ERK1/2, c-fos and c-jun were down-regulated too (t=0.776-1.192, all P<0.05). Conclusion: BDNF promotes the differentiation of iPSCs by activating Wnt/β-catenin and ERK/MAPK signal pathway, there should be cross-talk between the two signal pathways, and c-fos and c-jun may be common nuclear transcription factors. 目的： 探讨Wnt/β－连环素(β－catenin)和细胞外调节蛋白激酶/丝裂原活化蛋白激酶(ERK/MAPK)信号通路在脑源性神经营养因子(BDNF)促进诱导多能干细胞(iPSCs)分化为神经干细胞(NSCs)中的作用及相关机制。 方法： iPSCs传代培养、鉴定，BDNF和维甲酸(RA)诱导iPSCs分化为NSCs，流式细胞仪检测巢蛋白(Nestin)阳性细胞率。应用小分子RNA干扰技术(siRNA)分别沉默β－catenin及ERK1/2基因，并将iPSCs分为BDNF组(添加10 μg/L BDNF)、siRNA－ERK/BDNF组(转染siRNA－ERK并添加10 μg/L BDNF)、siRNA－β－catenin/BDNF组(转染siRNA－β－catenin并添加10 μg/L BDNF)及空白对照组分化培养，逆转录聚合酶链反应(RT－PCR)及Western blotting法检测各组Wnt/β－catenin和ERK/MAPK信号通路关键因子β－catenin、ERK1/2及下游转录因子c－fos、c－jun、c－myc的表达。组间两两比较用最小差值显著法进行分析。 结果： 免疫荧光染色显示iPSCs表达八聚体转录因子(Oct4)、胚胎干细胞转录蛋白(Sox2)及干细胞关键蛋白Nanog等多潜能标志物。流式细胞仪检测显示BDNF诱导iPSCs分化为NSCs的Nestin阳性细胞率为78.7%，而以RA为诱导剂的Nestin阳性细胞率为43.5%，不含诱导剂自然分化的Nestin阳性细胞率仅为7.8%。与对照组相比，BDNF组中β－catenin、ERK1/2、c－fos、c－jun及c－myc的mRNA表达明显上调(t＝2.805、2.318、2.255、1.799、1.582、1.663，均P<0.05)，蛋白表达也明显上调(t＝2.805、2.318、2.255、1.799、1.582、1.663，均P<0.05)。与BDNF组相比，siRNA－ERK/BDNF组中ERK1/2的mRNA及蛋白表达均明显下调(t＝1.917、2.042、1.673、1.540，均P<0.05)，c－fos、c－jun的mRNA及蛋白表达出现部分下调(t＝1.022、0.907、0.848、0.801，均P<0.05)，而β－catenin及c－myc的mRNA及蛋白表达无明显改变(t＝0.216、0.185、0.097、0.112，均P>0.05)；siRNA－β－catenin/BDNF组中β－catenin及c－myc的mRNA及蛋白表达明显下调(t＝3.104、2.774、2.235、1.911，均P<0.05)，而ERK1/2、c－fos及c－jun的mRNA及蛋白表达仅出现部分下调(t＝0.776～1.192，均P<0.05)。 结论： BDNF通过激活Wnt/β－catenin和ERK/MAPK信号通路促进iPSCs分化为NSCs，c－fos及c－jun是两条通路共同的核内转录因子，两条通路之间可能存在交互作用。.
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