血流切应力对血管内皮细胞PCP信号通路与初级纤毛发生的影响
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国家自然科学基金项目(31360278),贵州省科技厅联合基金项目[黔科合J字LKZ(2013)28号],遵义医学院博士启动基金(F566)


Effects of Fluid Shear Stress on PCP Signaling Pathway and Ciliogenesis in Vascular Endothelial Cells
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    摘要:

    目的 研究不同血流切应力(fluid shear stress, FSS)对内皮细胞平面细胞极性通路(planar cell polarity, PCP)的调节作用,进一步探讨FSS、PCP信号通路以及纤毛发生之间的关系。方法 建立可调控FSS的流体动力学细胞培养模型,qPCR以及免疫荧光检测不同FSS作用下PCP信号通路核心蛋白Dvl2及纤毛装配蛋白IFT88的mRNA表达与细胞定位以及两者的共定位,Western bolt (WB) 检测不同FSS作用18 h时Dvl2蛋白表达。结果 qPCR结果显示,与1.5 Pa比较,Dvl2 mRNA的表达量在0.1 Pa FSS作用6 h和18 h时均升高(P<0.05)、12 h时显著升高(P<0.01);IFT88 mRNA表达量在0.1 Pa FSS作用18 h时显著升高(P<0.01)。WB结果显示,与0 h比较,Dvl2蛋白的表达在0.1 Pa FSS作用18 h时升高(P<0.05),在1.5 Pa FSS作用18 h时显著降低(P<0.05);与1.5 Pa FSS比较,Dvl2 蛋白的表达在0.1 Pa FSS作用18 h时升高(P<0.05)。免疫荧光结果显示,Dvl2蛋白阳性表达随 FSS作用时间的增加增多,且逐渐聚集于细胞核周边一点;IFT88蛋白阳性表达在0.1 Pa FSS作用下逐渐由细胞核向细胞质转移并聚集为一点,1.5 Pa FSS作用下逐渐减少且解聚;蛋白Dvl2、IFT88在0.1 Pa FSS作用下均定位于细胞的同一位置,在1.5 Pa FSS作用18 h内均定位于细胞的同一位置,18 h后由于蛋白IFT88发生解聚,未观察到共定位。结论 层流 FSS作用能够抑制PCP信号通路的转导并阻碍纤毛发生,低 FSS促进其转导,且PCP信号通路可能通过Dvl2调控FSS诱导的初级纤毛发生。

    Abstract:

    Objective To investigate the effect of different fluid shear stress (FSS) on the regulation of planar cell polarity (PCP) signaling, and further to explore the relationship among FSS, PCP signaling pathway and ciliogenesis. Methods The hydrodynamic cell model of adjustable FSS was established. qPCR and immunofluorescence were used to detect the mRNA expression of PCP signaling pathway core protein Dvl2 and cilia assembly protein IFT88, cell targeting and co-localization under different FSS. Western blot (WB) was used to detect the protein expression of Dvl2 at 18 h under different FSS. Results The qPCR result showed that compared with 1.5 Pa FSS, under 0.1 Pa FSS, the mRNA expression of Dvl2 was higher at 6 h and 18 h (P<0.05), significantly higher at 12 h (P<0.01); the mRNA expression of IFT88 was significantly higher at 18 h (P<0.01). The WB result showed that compared with 0 h, under 0.1 Pa FSS, the protein expression of Dvl2 was higher at 18 h (P<0.05), significantly lower under 1.5 Pa FSS (P<0.01); compared with 1.5 Pa FSS, the protein expression of Dvl2 was higher at 18 h under 0.1 Pa FSS (P<0.05). The immunofluorescence result showed that the positive expression of Dvl2 increased with the loading time on FSS increasing, and gradually aggregated at a point around the nucleus; the positive expression of IFT88 was gradually transferred from the nucleus to the cytoplasm and aggregated at a point under 0.1 Pa FSS, and gradually decreased and depolymerized under 1.5 Pa FSS. Protein Dvl2 and IFT88 were located in the same position in cells under 0.1 Pa FSS and before 18 h under 1.5 Pa FSS, and colocalization of proteins Dvl2 and IFT88 was not observed after 18 h under 1.5 Pa FSS due to IFT88 depolymerization. Conclusions Laminar FSS played an inhibition on the transduction of PCP signaling pathway and a hindrance on the process of ciliogenesis, while low FSS played a promotion on the transduction. PCP signaling pathway might regulate FSS-induced ciliogenesis by Dvl2.

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刘月华,生燕,欧刚卫,生欣.血流切应力对血管内皮细胞PCP信号通路与初级纤毛发生的影响[J].医用生物力学,2019,34(1):64-68

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  • 收稿日期:2018-01-30
  • 最后修改日期:2018-03-17
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  • 在线发布日期: 2019-03-01
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