podocin基因敲低对小鼠足细胞nephrin和α-actinin表达与分布的影响

Influence of the sequence specific knockdown of podocin mRNA on the expression of nephrin and α actinin in mouse podocyte

目的研究足细胞分子podocin、nephrin和α-actinin之间的相互作用和联系。方法将针对podocin分子设计的特异RNA干扰(RNAi)质粒导入体外培养的小鼠足细胞系(MPC5);应用免疫荧光染色及双标记在共聚焦显微镜下观察nephrin、podocin和α-actinin的分布方式;半定量RT-PCR和免疫蛋白印迹检测podocin、nephrin,α-actinin-4及内参照GAPDH/β-actin在mRNA水平和蛋白水平的表达量。结果(1)干扰组podocin的荧光强度显著低于对照组,其mRNA和蛋白的表达量分别下降了65%和89%。免疫双标记显示podocin的分布发生了明显变化:对照组podocin不但分布在胞核的周围,而且在胞浆呈放射性沿着细胞骨架分布,以及主要在胞膜上呈连续性的线状分布;干扰组podocin呈点状分布在胞核周围。(2)干扰组nephrin的荧光强度亦明显减低,其mRNA及蛋白的表达量分别减少了70%和78%。免疫双标记显示nephrin的分布也发生了显著变化:干扰组nephrin亦呈点状分布在胞核周围;在对照组,nephrin和podocin的分布方式相同。(3)干扰组和对照组α-actinin的分布及在mRNA和蛋白水平上的表达量无明显差异,呈细丝状均匀分布于胞质内,亦呈放射性分布于足细胞伸出的突起中。结论通过RNAi成功地使MPC5的podocin表达下调。Podocin和nephrin分子关系紧密,可能存在着?

Objective To study the molecular interaction among podocin,nephrin and α actinin. Methods Firstly, the recombinant RNA interference (RNAi) plasmid specifically targeting podocin mRNA was tansfected into mouse podocyte clone(MPC5). The distribution levels of podocin, nephrin and α actinin were revealed by immunofluorescence staining, and double immunolabeling under laser scanning confocal microscope. The mRNA and protein expression of nephrin, podocin, α actinin and GAPDH/β actin were detected by semi quantitative RT PCR and Western blotting. Results The fluorescence intensity of podocin and nephrin was significantly lower than that in control group. The reductions of the mRNA expression in podocin and nephrin were apparently detected by about 65%and 70%, respectively. The protein expression levels of podocin and nephrin decreased significantly by about 89%and 78%, respectively. The distribution changes of podocin and nephrin were found as compared to control groups. The staining of podocin and nephrin in control groups was distributed around nuclei and mainly on the cell membrane surface in a filamentous pattern, while their staining in interference group was predominantly located around nuclei. The mRNA and protein expression, and the distribution of α actinin showed no difference from those in the control group. They were predominantly localized in the cytoplasm with a filamentous pattern and also extended to processes. Conclusion The expression of podocin is successfully suppressed with RNAi. Podocin may not interact directly with α actinin, but with nephrin to some extent.