T_3对人神经干细胞分化为少突胶质细胞的影响

EFFECTS OF T_3 ON DIFFERENTIATION OF HUMAN NEURAL STEM CELLS TO OLIGODENDROCYTE

目的 以体外诱导生成的人神经干细胞 (NSCs)为模型 ,研究甲状腺激素 (TH)对其分化的影响。 方法 无菌状态下取因治疗目的而终止妊娠的胎龄 10～ 2 2周的人胎大脑半球 ,机械法分散细胞后 ,以 10 5个 ml细胞密度接种于含N 2配方的DMEM/F12培养基中 ,同时添加表皮生长因子 (EGF ,2 0 μg L)和 /或碱性成纤维生长因子(bFGF ,2 0 μg L)。采用自然分化以及T3诱导的方法诱导分化 ,免疫组织化学方法鉴定分化后的细胞类型 ,抗体分别为NF 2 0 0、GFAP、Gal C ,并采用抗MBP、O4、A2B5抗体鉴别少突胶质细胞发育的不同阶段。 结果 T3有助于向胶质细胞方向发展 ,包括少突与星形胶质细胞 ,MPB阳性细胞比例超过 80 % ,尤其在EGF +T3组 ,这种现象更为突出。 结论 NSCs的定时分化是内外源信号共同作用的结果。TH就是这样一种信号 ,激活“生物钟”机制的效应组件 。

Objective The aim of the study was to assess the human neural stem cells (NSCs) induced by mitogens in vitro and investigate effects of TH(T 3) on differentiation. Methods The cerebral hemispheres of human fetal at 10-12 weeks of gestation were minced and mechanically dissociated. Cells were seeded at a concentration of 10 5 per ml into defined medium that consisted of DMEM/F12 Epidermal growth factor (EGF,20?μg/L) and basic fibroblast growth factor (bFGF,20?μg/L) were added respectively as mitogens for 7 days. In some cases, triiodothyronine (T 3, 30?μg/L) was added through the culture period. Upon differentiation, the cell cultures were exposured to a substrate and treated with or without T 3 after removing the mitogens. To detect the phenotypes of differentiated cells, immunochemistry staining was performed with the antibody to NF 200, GFAP, MBP and Gal C. To recognize the different stages of oligodendrocytes development, the antibodies O4 and A2B5 were used. The proportion of each neural cell type was determined by counting positive cells in standardized fields at 40× magnifications. Results NSCs induced by EGF and/or bFGF grew in culture as free floating spheres(neurospheres) and were immunoreactive for the intermediate filament nestin. After removing EGF and bFGF, the cells cease mitosis and can be induced to differentiate into neurons, astrocytes, and oligodendrocytes regardless of whether T 3 was presented. T 3 favored an neuroglia cell fate especially when combining EGF with T 3 and differentiated cells appeared more early when added of T 3.MBP positive cell is over 80%. The O4 and A2B5 positive cells can be observed at early stage of differentiation only after process grew from neurospheres after adhesion to certain substrate, which indicated that the neuronal network could promote the maturity of oligodendrocyte.Discussion The timing of NSCs differentiation depends on both intracellular mechanisms and extracellular signals. TH is such a signal to activate the effector component of a “clock” mechanism that induces oligodendrocyte differentiation after a limited number of cell divisions.[