EGF和FGF-2促神经干细胞增殖与分化研究

The study on EGF and FGF-2 in promoting the proliferation and differentiation of NSCs

目的 建立人胚胎大脑神经干细胞的分离培养方法并观察在不同诱导因子作用下细胞分化的生物学特性。方法 将经培养的人胚胎大脑神经干细胞分为4组,在不同培养液中培养、分化:(1)DMEM/F-12标准培养液组;(2)标准培养液+表皮生长因子(EGF)组;(3)标准培养液+碱性成纤维细胞生长因子-2(FGF-2)组;(4)标准培养液+表皮生长因子+碱性成纤维细胞生长因子-组。用定量RT-PCR方法及免疫组织化学染色方法鉴定神经干细胞的原始特性、端粒酶活性以及不同诱导因子对神经干细胞分化特性的影响。结果 于培养第6d,标准培养液中的脑细胞死亡;而在加入EGF和FGF-2的培养液中则形成神经干细胞球体,而且增殖细胞核抗原染色和神经干细胞巢蛋白表达均呈阳性反应;其端粒酶活性为63％,低于瘤细胞而高于正常脑组织。神经干细胞分化后经胶质纤维酸性蛋白、神经微丝、突触素、微管相关蛋白-2及神经细胞核抗原染色显示,分化的神经元最高值达(18±2.5)％。不同诱导因子对不同传代的细胞球体内神经干细胞的促增殖作用差异有显著性意义(均P<0.05)。结论 EGF和EGF-2均具有促进神经干细胞存活、增殖和分化的作用。

Objective To establish the isolation and culture technique of human fetal brain neural stem cells (NSCs) and study its biological characteristics in differentiation with induction factors of generated cells. Methods The human fetal brain NSCs were cultured and differentiated in: 1)DMEM/F-12 culture solution (group 1, control group); 2)DMEM/F-12+epidermal growth factor (EGF, group 2);3)DMEM/F-12+basic fibroblast growth factor-2 (FGF-2, group 3) and 4)DMEM/F-12+EFG+FGF-2 (group 4). The primitive character of NSCs, activity of telomerase and effect of induction factor on differentiation were identified by reverse transcriptive polymerase chain reaction (RT-PCR) and immunohistochemical staining. Results The NSCs died in 6 d in DMEM/F-12 culture solution. The NSCs neurospheres were formed in the presence of EGF and FGF-2 in culture. The positive reactions were showed in nucleus staining of proliferated cells and NSCs nestin expression. The telomerase activity of the proliferated cells was 63% which was lower than that in tumor cells and higher than normal brain cells. Positive reactions of differentiatetb NSCs were showed irr the staining of glial fibrillary acid protein, microtubule associated protein-2, neurofilament, synaptophysin and neuron nucleoantigen. The maximal differentiation rate of neurons was (18.6±2.5)%. The differentiation promoting effects on different generations of NSCs sphere with various induction factors were different significantly (P<0.05). Conclusion It demonstrates that both EFG and FGF-2 possess the promoting effects for survival, proliferation and differentiation on NSCs.