来源于大鼠脊髓星形胶质细胞的神经干细胞的增殖分化特性

Differentiation and proliferation patterns of neural stem cells of astrocyte origin in the rat spinal cord

目的:通过研究碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)在A-NSC培养中的作用来观察其增殖特性;通过添加血小板生长因子PDGF-AA作为诱导因子来研究A-NSC分化的特性。方法:将A-NSC分四组:bFGF+EGF添加组,bFGF添加组,EGF添加组和对照组进行培养,通过形态观察和流式细胞仪检测细胞周期。同时添加PDGF-AA诱导A-NSC分化,以自然分化为对照组,通过免疫荧光染色观察比较其各类细胞分化比例的差异。结果:可观察到A-NSC在bFGF添加组和bFGF+EGF添加组有正常的分裂周期,而EGF添加组和对照组的细胞几乎都停滞在G0/G1期,显示其增殖受到阻滞;诱导实验检测到对照组细胞的分化比例分别为神经元(89.0±4.2)%,星形胶质细胞(4.6±3.2)%,少突胶质细胞(7.6%±2.5%),具有很高的神经元分化比例;诱导组细胞的分化比例分别为神经元(80.6±4.1)%,星形胶质细胞(3.0±1.3)%,少突胶质细胞(20.6±1.8)%,诱导组分化成少突胶质细胞的能力有显著提高(P<0.001)。结论:在A-NSC的生长过程中,bFGF对A-NSC的增殖起重要作用,在本实验条件下A-NSC倾向于向神经元分化,同时PDGF-AA能够显著诱导A-NSC分化成少突胶质细胞。

Objective： To find the proliferation pattern of astrocyte-origin neural stem cells by investigating the effects of basic fibroblast growth factor（bFGF） and epidermal growth factor（EGF） on cell growth and proliferation;To find the differentiation pattern of A-NSC by adding platelet-derived growth factor（PDGF-AA） as inducing factor.Methods：In this study,four groups of A-NSC were cultured with the addition of either bFGF or EGF,or both,no addition of these two growth factors as control.The cellular growth and cycle were checked though microscopic observation and flow cytometry analysis;PDGF-AA was used as inducing factor during A-NSC differentiation to observe and compare the percentage of various cell types through immunofluorescence staining,natural differentiation as control.Results： It showed that with bFGF＋EGF or bFGF treatments,A-NSC could proliferate and have whole cell cycle;with only EGF or without two growth factors treatments,the proliferation of cells was blocked;flow cytometry data showed that almost all the cells stayed at G0/G1 stage.In the induction study,data showed that control group had the percentage rate as follow：Tuj1 positive neurons with high percentage for 89±4.2%,GFAP positive astrocyte 4.6±3.2%,O4 positive oligodendrocyte（OLs） 7.6±2.5%;the PDGF-AA induced group for neuron,astrolyte and oligoendrocyte were 80.6±4.1%,3.0±1.3% and 20.6±1.8% respectively.The ability of cells in induced group differentiating into O4 positive OLs increased significantly（P0.001）.Conclusion： In this study,bFGF is important to A-NSC in cell growth and proliferation,A-NSC is prone to differentiate into the neurons;PDGF-AA could significantly induce A-NSC into higher rate of OLs.