Podocalyxin基因沉默对足细胞结构和功能的影响

Podocalyxin gene silencing on structure and function of podocyte

目的:探讨siRNA敲低podocalyxin(PODXL、足糖萼)基因对足细胞结构、功能的影响及机制。方法:采用RPMI 1640培养基,在33℃培养永生化小鼠足细胞系,分化成熟后分为5组:空白组(Vehicle)、模拟组(Mock)、阴性对照组(Sh-nc)、siRNA-PODXL组(Si)、ezrin抑制组(NSC305787)。用鬼笔环肽行足细胞骨架蛋白Factin染色,共聚焦荧光倒置显微镜下观察细胞骨架排列;划痕愈合实验、Transwell实验检测细胞的迁移能力;流式细胞仪检测足细胞凋亡; Western印迹法和RT-qPCR检测PODXL和p-ezrin表达水平。结果:(1)共聚焦荧光倒置显微镜下观察结果显示:空白组足细胞骨架肌纤维排列有序,荧光显示清晰; Si组、ezrin抑制组足细胞微丝解聚、胞体回缩,骨架排列紊乱,荧光明显模糊减弱,组间比较差异有统计学意义(P<0. 05); Mock及Sh-nc组与空白组无明显差异;(2)划痕愈合实验、Transwell实验结果显示:PODXL敲低、抑制ezrin磷酸化后足细胞迁移较其他三组减慢;(3)流式细胞仪结果显示:PODXL敲低、抑制ezrin磷酸化后足细胞凋亡率增加;(4) western印迹法和RT-qPCR检测结果显示:与正常组相比,Si组PODXL表达明显下调,同时p-ezrin表达下调,Mock及Sh-nc组与空白组无明显差异;(5) G-LISA法检测细胞中RhoA活性,结果显示:Si组、ezrin抑制组与正常组相比RhoA活性明显下降,Mock及Sh-nc组与空白组无明显差异。结论:敲低PODXL基因或抑制ezrin磷酸化后,p-ezrin表达下调、RhoA活性降低,导致足细胞骨架结构紊乱、促进足细胞凋亡。表明PODXL基因敲低后引起足细胞损伤可能与p-ezrin、RhoA信号通路有关。

Objective: To explore effects of siRNA knockdown podocalyxin on the structure and function of podocytes. Methodology: Podocytes were cultured in RPMI 1640 medium at 33 ℃,and switch to 37 ℃ for differentiation and maturation.Cells were then divided into 5 groups: Vehicle,Mock,Sh-scramble control( Sh-nc),siRNA-PODXL( Si),and ezrin inhibition group( NSC305787). Fluorescence microscopy was used to evaluate the changes in cell structure and actin cytoskeleton after phalloidin staining of F-actin.The wound healing experiment and Transwell test were used to assess cell migration,and flow cytometry was used to detect apoptosis of podocyte.Western blot and RT-qPCR were used to quantify change of PODXL and p-ezrin gene and protein. Results:( 1) Cytoskeleton of podocyte in Vehicle group was normal,while in the cells with PODXL knockdown or with ezrin inhibited,F-actin stress fibers were rearranged and lost( P<0. 05).There was no significant change of F-actin stress fibers in the Mock and Sh-nc groups compared with Vehicle group.( 2) The wound healing and Transwell experiments both showed that migration of podocyte in the Si group,ezrin inhibition group were slower than that of the other three groups.( 3) Flow cytometry showed that apoptosis increased in cells with PODXL knockdown or ezrin inhibition.( 4) Western blotting and RT-PCR analysis showed that PODXL was successfully knocked down in Si group,accompanied by downregulation of p-ezrin but not total ezrin compared with cells in the Vehicle group;( 5) Compared with Vehicle group,the activity of Rho A decreased significantly in Si group and ezrin inhibition group,but there was no significant change of Rho A activity in Mock and Sh-nc groups compared with vehicle group. Conclusion:PODXL plays a key role on podocyte cytoskeletons and survival,and maintains the level of podocyte p-ezrin which may be crucial for podocyte cytoskeletons and survival.