碱性成纤维细胞生长因子-壳聚糖载体诱导神经干细胞向神经元分化的机制

摘要

Objective To investigate the potential mechanism of basic fibroblast growth factor (bFGF)-chitosan carrier to induce neural stem cells to differentiate into neurons. Methods After purification, the neural stem cells were cocultured with chitosan, soluble bFGF and bFGF-chitosan carrier. Three hours, twenty-four hours, three days and seven days after induction, immunofluorescence staining of Nestin, beta tubulin III, microtubule-associated protein-2 (MAP2), and fibroblast growth factor receptor 1 (FGFR1) were used to observe the expres-sion of FGFR1;real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to detect RNA and protein level changes after induction. Results Three hours after induction, there was no significant difference in the expression of FGFR1 among three groups. Twenty-four hours after induction, the expression level of FGFR1 was significantly higher in the bFGF-chitosan carrier group than in the chitosan group and the soluble bFGF group (P<0.001);three days and seven days after induction, the expression of FGFR1 decreased significantly in the chitosan group and soluble bFGF group (P<0.001), however, it was still higher in the bFGF-chitosan carrier group;moreover, the expression of genes associated with the pathway of extracellular regulated protein kinases/mitogen activated protein ki-nase (Erk/MAPK) was significantly higher in the bFGF-chitosan carrier group than in the chitosan group and soluble bFGF group (P<0.001). Conclusion bFGF-chitosan carrier might upregulate the expression of FGFR1, then activate Erk/MAPK signal pathways, and finally promote the differentiation of neural stem cells into neurons.%目的：探讨碱性成纤维细胞生长因子(bFGF)-壳聚糖载体诱导神经干细胞高比例向神经元分化的潜在机制。方法纯化后神经干细胞分别与单纯壳聚糖、可溶性bFGF和bFGF-壳聚糖载体共培养。在共培养3 h、24 h、3 d和7 d后，Nestin、β-Ⅲtu-bulin、微管相关蛋白2(MAP2)和成纤维细胞生长因子受体1(FGFR1)的免疫荧光双标记观察受体的表达情况；实时荧光定量PCR (qRT-PCR)及Western blotting分别检测不同时间点神经干细胞诱导后相关基因的RNA水平变化。结果诱导后3 h，FGFR1的表达量在三组中无明显差异；诱导后24 h，bFGF-壳聚糖载体组FGFR1的表达量显著高于单纯壳聚糖组和可溶性bFGF组(P<0.001)；诱导后3 d和7 d，FGFR1的表达量在单纯壳聚糖组和可溶性bFGF组显著减少(P<0.001)，而在bFGF-壳聚糖载体组仍维持较高水平；qPT-PCR及Western blotting结果显示，bFGF-壳聚糖载体组中与Erk/MAPK信号通路相关基因的表达水平显著高于单纯壳聚糖组和可溶性bFGF组(P<0.001)。结论 bFGF-壳聚糖载体通过缓释bFGF，可能上调FGFR1的表达，进而激活Erk/MAPK信号通路，促进神经干细胞向神经元的分化。