蔡明, 陆耀飞, 娄淑杰*
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PI3-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% O2、1% O2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PI3-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PI3-K信号通路在此过程中发挥主要作用。
[Effects of hypoxia conditioned medium of cerebral cortex cells on the differentiation of neural stem cells and related signal pathways.] [Article in Chinese]
CAI Ming, LU Yao-Fei, LOU Shu-Jie*
School of Kinesiology, Shanghai University of Sport, Shanghai 200438，China
The aim of this study was to investigate the effects of hypoxia conditioned medium (HCM) of cerebral cortex cells on the differentiation of neural stem cells (NSCs) and to clarify the signal transduction mechanism. The cerebral cortex cells from newborn SD rats were primarily cultured for 5 d, and then the cells were cultured in environments of 4% O2, 1% O2 and normal oxygen concentration, respectively, for 6 h. The culture mediums were collected and centrifuged as the HCM and normoxia conditioned medium (NCM). The neurospheres of NSCs were obtained from the rat cerebral cortex by suspending culture. Immunohistochemical staining was used after adherence culture for 48 h to identify neurons and astrocytes in the progeny of NSCs. LY294002, a PI3-K inhibitor, and SP600125, a JNK inhibitor, were added into the HCM to culture NSCs for 48 h. The results showed that NSCs in the cerebral cortex could differentiate into β-Tubulin III immunoreactive neurons and GFAP immunoreactive astrocytes in three conditioned mediums, and the neurons proportion in progeny of NSCs was higher than astrocytes in all three groups. The proportion of neurons in 4% HCM was higher than that in NCM (P < 0.01). But the proportion of neurons in 1% HCM was less than that in NCM (P < 0.01). Both LY294002 and SP600125 inhibited NSCs to differentiate into high proportion neurons induced by 4% HCM (P < 0.01), but the inhibitory effect of LY294002 was stronger than that of SP600125 (P < 0.01). In conclusion, 4% HCM can induce NSCs to differentiate into more neurons mainly through the PI3-K pathway.
通讯作者：娄淑杰 E-mail: firstname.lastname@example.org
蔡明, 陆耀飞, 娄淑杰. 大脑皮层细胞低氧条件液诱导神经干细胞分化及其可能的信号转导通路[J]. 生理学报 2013; 65 (5): 497-504.
CAI Ming, LU Yao-Fei, LOU Shu-Jie. [Effects of hypoxia conditioned medium of cerebral cortex cells on the differentiation of neural stem cells and related signal pathways.] [Article in Chinese]. Acta Physiol Sin 2013; 65 (5): 497-504 (in Chinese with English abstract).