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  

Mechanism of Basic Fibroblast Growth Factor-chitosan Carrier Inducing Neural Stem Cells to Differentiate into Neurons

作　　者：段红梅[1,2] 王聪[2] 杨朝阳[1,2] 郝鹏[2] 尚俊奎 李晓光[1,2] 

机构地区：[1]北京航空航天大学生物与医学工程学院,北京市100191 [2]首都医科大学神经生物学系,北京市100069

出　　处：《中国康复理论与实践》2016年第5期528-534,共7页Chinese Journal of Rehabilitation Theory and Practice

基　　金：国家“863”计划项目(No.2012AA020506);国家自然科学基金面上项目(No.31271037);国家自然科学基金国际合作与交流项目(No.31320103903);“十二五”国家科技支撑计划项目(No.2012BAI17B04);高等学校全国优秀博士学位论文作者专项资金资助项目(No.201356);国家国际科技合作专项项目(No.2014DFA30640);国家自然科学基金委员会资助项目(No.31130022)

摘　　要：目的探讨碱性成纤维细胞生长因子(b FGF)-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b FGF和b FGF-壳聚糖载体共培养。在共培养3 h、24 h、3 d和7 d后,Nestin、β-Ⅲtubulin、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b FGF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b FGF组显著减少(P<0.001),而在bFGF-壳聚糖载体组仍维持较高水平;q PT-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。Objective To investigate the potential mechanism of basic fibroblast growth factor（b FGF）-chitosan carrier to induce neural stem cells to differentiate into neurons. Methods After purification, the neural stem cells were cocultured with chitosan, soluble b FGF and b FGF-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 expression of FGFR1; real-time fluorescent quantitative polymerase chain reaction（q RT-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 b FGF-chitosan carrier group than in the chitosan group and the soluble b FGF group（P0.001）; three days and seven days after induction, the expression of FGFR1 decreased significantly in the chitosan group and soluble b FGF group（P0.001）, however, it was still higher in the b FGF-chitosan carrier group; moreover, the expression of genes associated with the pathway of extracellular regulated protein kinases/mitogen activated protein kinase（Erk/MAPK） was significantly higher in the bFGF- chitosan carrier group than in the chitosan group and soluble bFGF group（P0.001）. 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.

关 键 词：碱性成纤维细胞生长因子 壳聚糖 神经元 神经干细胞 分化 大鼠 

分 类 号：R644[医药卫生—外科学]