缺氧预处理对神经干细胞间隙连接蛋白的表达及半通道功能的影响

目的:检测缺氧预处理对神经干细胞间隙连接蛋白的表达情况及半通道功能的影响,为探究缺氧预处理后神经干细胞间细胞通讯提供理论依据。方法:采用免疫组织化学方法检测对照组及缺氧预处理1小时组、缺氧预处理4小时组、缺氧预处理8小时组、缺氧预处理12小时组神经干细胞中cx43、cx36、cx47的表达情况。通过激光共聚焦激光扫描显微镜测量钙黄绿素滤过情况以评估半通道功能的变化。结果:与对照组(1.03±0.14%)比较,在0.5%氧气条件下暴露8小时(3.05±0.40%)和12小时(5.51±0.31%)的神经干细胞坏死率较显著升高(P0.05)。与对照组相比,缺氧预处理4、8、12小时组神经干细胞中cx43、cx36、cx47的表达显著增加,且随着缺氧预处理时间的延长而显著增加(P0.05)。与对照组及加入半通道阻滞剂组相比,缺氧预处理组半通道滤过功能显著增强(P0.05)。结论:缺氧预处理可以以时间依赖性方式提高神经干细胞间隙连接蛋白的表达和半通道滤过功能。神经干细胞暴露于低氧环境而不引起坏死显著增加的最佳时间为4小时。

Hypoxic Preconditioning Increases the Expression of Connexins and the Hemichannel Filtration Function

ABSTRACT Objective: Determine the expression of gap junction protein and the changes of hemichannel function after hypoxia preconditioning of neural stem cells, which provided a theoretical basis for exploring the communication between neural stem cells after hypoxia preconditioning. Create a standardized preconditioning procedure and reproducibly expose the NSCs to hypoxia.Explore the best time for neural stem cells to be exposed to hypoxia without increasing necrosis. Methods: The expression of cx43, cx36, and cx47 in the neural stem cells of the control group and the hypoxia- preconditioned neurons were determined by immunohistochemical method at 1 h, 4 h, 8 h, and 12 h, respectively. The calcein dye efflux experiments was observed by laser confocal laser scanning microscopy to observe changes in hemichannell function. Results: Compared with the control group (1.03+0.14%), the necrosis rate of neural stem cells in 8 hours (3.05+0.40%) and 12 hours (5.51+0.31%) exposed to 0.5% oxygen was significantly higher (P<0.05). Compared with the control group, the expression of cx43, Cx36 and Cx47 in neural stem cells increased significantly after hypoxic preconditioning for 4, 8 and 12 hours (P<0.05). Hypoxia pretreatment group had significantly enhanced hemichannel filtration function compared with the control group and the hemichannel blocker group. Conclusion: Hypoxic preconditioning can improve the expression of connexin and the half-channel filtration function of neural stem cells in a time-dependent manner. The optimal time for neural stem cells to be exposed to hypoxia without causing significant increase in necrosis is 4 hours.