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本研究旨在分析大鼠大脑皮层细胞低氧条件液对神经干细胞(neural stem cells,NSCs)分化的影响,并探讨P13-K和JNK两条信号通路在此过程中的作用。原代培养出生后24 h内SD大鼠大脑皮层细胞5 d后,全量换液并分别在4%O_2、1%O_2和常氧浓度环境下继续培养细胞6 h以制备低氧条件液和常氧条件液。用三种条件液及结合使用P13-K、JNK信号通路抑制剂LY294002和SP600125培养NSCs,使用免疫荧光双标法鉴定NSCs后代细胞表型,并计算每种表型细胞在后代细胞中所占比例,以分析不同条件下NSCs的分化情况。结果显示,在三种条件液培养下NSCs分化出神经元的比例均高于星形胶质细胞的比例;与常氧条件液组比较,4%低氧条件液促进了NSCs向神经元方向分化(P<0.01),而1%低氧条件液则抑制了NSCs向神经元方向分化(P<0.01)。在4%低氧条件液中使用LY294002和SP600125均抑制了NSCs向神经元方向分化(P<0.01),且LY294002抑制作用较SP600125大(P<0.01)。以上结果提示,4%低氧条件液能促进大脑皮层NSCs向神经元方向分化,且P13-K信号通路在此过程中发挥主要作用。
The purpose of this study was to investigate the effects of hypoxic conditions on the differentiation of neural stem cells (NSCs) in rat cerebral cortical cells and to explore the role of P13-K and JNK signaling in this process. Primary culture of SD rats within 24 h after birth, the cerebral cortex cells for 5 d, the full amount of liquid exchange, respectively, and 4% O2, 1% O2 and normoxic concentrations continued to culture cells for 6 h to prepare hypoxic condition fluid and often Oxygen conditions. NSCs were cultured with three conditions and P13-K combined with JNK signaling inhibitors LY294002 and SP600125. The phenotypes of progeny of NSCs were identified by immunofluorescence double-labeling and the proportion of each phenotype in progeny cells was calculated To analyze the differentiation of NSCs under different conditions. The results showed that the proportion of NSCs differentiated into neurons under three conditions was higher than that of astrocytes. Compared with the normoxemia group, 4% hypoxic conditions promoted the differentiation of NSCs into neurons (P <0.01), while 1% hypoxic condition inhibited the differentiation of NSCs into neurons (P <0.01). The use of LY294002 and SP600125 in 4% hypoxic conditions inhibited the differentiation of NSCs into neurons (P <0.01), and the inhibitory effect of LY294002 was greater than that of SP600125 (P <0.01). The above results suggest that 4% hypoxic condition fluid can promote the differentiation of NSCs into neurons in cerebral cortex, and the P13-K signaling pathway plays a major role in this process.