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目的:观察常氧和低氧环境下大鼠大脑皮层神经干细胞(NSCS)体外分化情况,并比较PI3K/Akt、JNK和Notch三条信号通路在不同氧环境下神经干细胞分化时所发挥作用的异同。方法:将体外悬浮培养48 h的神经干细胞转移至4%低氧和常氧中贴壁培养。更换培养基诱导分化,同时加入LY294002(PI3-K/Akt抑制剂)、SP600125(JNK抑制剂)和DAPT(Notch抑制剂),48 h后行β-微管蛋白Ⅲ(β-TubulinIII)和胶质纤维酸性蛋白(GFAP)免疫细胞荧光染色。对神经元和星形胶质细胞进行计数并分别计算各占的比例。结果:(1)低氧对照组神经元所占比例高于常氧对照组,星形胶质细胞低于常氧对照组,均具有统计学差异(P<0.01)。(2)在常氧环境下,LY294002组神经元所占比例显著低于常氧DMSO对照组(P<0.01),星形胶质细胞所占比例显著高于常氧DMSO对照组(P<0.01);和常氧DMSO组相比,SP600125组和DAPT组神经元和星形胶质细胞所占比例均无显著差异。(3)在低氧环境下,和低氧DMSO组比较,LY294002组和SP600125组神经元和星形胶质细胞所占比例均无显著性差异;DAPT组神经元所占比例显著高于常氧DMSO对照组(P<0.01),星形胶质细胞所占比例低于常氧DMSO对照组,无显著性差异。结论:PI3K/Akt介导了常氧条件下神经干细胞向神经元方向的分化;低氧可能通过抑制Notch信号通路促进神经干细胞向神经元方向分化。
OBJECTIVE: To observe the differentiation of neural stem cells (NSCs) in vitro and in vitro in rats under hypoxia and hypoxia, and to compare the roles of PI3K / Akt, JNK and Notch in the differentiation of neural stem cells in different oxygen environments. Methods: Neural stem cells cultured in suspension for 48 h were transferred to 4% hypoxic and normoxia cultures. The medium was changed to induce differentiation. At the same time, LY294002 (PI3-K / Akt inhibitor), SP600125 (JNK inhibitor) and DAPT (Notch inhibitor) were added and 48 h later, β-TubulinIII Fibroblast acidic protein (GFAP) immunofluorescence staining. Neurons and astrocytes were counted and the proportions were calculated separately. Results: (1) The proportion of neurons in hypoxic control group was higher than that of normoxia control group and astrocyte was lower than that of normoxia control group (P <0.01). (2) Under normoxia, the proportion of neurons in LY294002 group was significantly lower than that in normoxic DMSO control group (P <0.01), the proportion of astrocytes was significantly higher than that in normoxia DMSO control group (P <0.01) ). Compared with normoxic DMSO group, the proportion of neurons and astrocytes in SP600125 group and DAPT group showed no significant difference. (3) Compared with hypoxia DMSO group, the proportion of neurons and astrocytes in LY294002 group and SP600125 group had no significant difference in hypoxic environment; the proportion of neurons in DAPT group was significantly higher than that of normoxia group DMSO control group (P <0.01), the proportion of astrocytes was lower than that of normoxic DMSO control group, no significant difference. Conclusion: PI3K / Akt mediates the differentiation of neural stem cells into neurons under normoxia. Hypoxia may promote the differentiation of neural stem cells into neurons by inhibiting Notch signaling pathway.