初级纤毛细胞外氧感受器功能和机制研究

摘要

目的:探索初级纤毛是否为PC12细胞的细胞外氧感受器.方法:通过小干扰RNA(siRNA)干扰细胞纤毛内转运蛋白88的表达抑制体外培养PC12细胞初级纤毛的发生;免疫荧光染色法观察缺氧诱导因子1α(HIF-1α)、核因子E2相关因子2(Nrf2)核转位和PC12细胞初级纤毛生长情况;实时定量RT-PCR法检测HIF-1α、Nrf2、血管内皮生长因子(VEGF)和超氧化物歧化酶(SOD)基因表达.结果:siRNA干扰组细胞的初级纤毛被抑制.与常氧对照组比较,缺氧对照组和阴性对照组细胞的HIF-1α和Nrf2发生了明显的核转位,细胞内HIF-1α、Nrf2、VEGF基因表达量增加,SOD表达量下降;siRNA干扰组细胞在缺氧状态下HIF-1α和Nrf2未发生核转位,细胞内HIF-1α、Nrf2、VEGF 基因表达受到抑制,而SOD 表达量升高.结论:PC12细胞的初级纤毛可能具有细胞外氧感受器的作用,可以感知细胞外环境中的缺氧和氧化应激状态,并通过一定途径将缺氧信号传导至细胞内,进而启动细胞抗缺氧机制.%Objective: To investigate the function of primary cilium as an oxygen sensor in PC12 cells. Methods: The PC12 cells were transfected with IFT88 siRNA.The nuclear translocation of hypoxia inducible factor-1α (HIF-1α), nuclear factor erythroid-2 related factor 2 ( Nrf2 ), and ciliogenesis were observed by immunofluorescence staining; and the mRNA expressions of HIF-1α, Nrf2, vascular endothelial growth factor (VEGF) and superoxide dismutase (SOD) were detected by real-time RT-PCR. Results: The ciliogenesis was inhibited in PC12 cells transfected with IFT88 siRNA. In hypoxia group and scramble control group,nuclear translocations of HIF-1α and Nrf2 were observed and mRNA expressions of HIF-1α, Nrf2, VEGF were increased,and those of SOD were decreased. While in PC12 cells transfected with IFT88 siRNA, nuclear translocations of HIF-1α and Nrf2 were not observed, and mRNA expressions of HIF-1α, Nrf2, VEGF were inhibited, and mRNA expression of SOD was increased. Conclusion:Primary cilia may act as an oxygen sensor to transfer the information related to hypoxia and oxidative stress into cells,activating intracellular defense mechanism against the hypoxic injuries.