【摘要】 目的:研究小干扰RNA(siRNA)干扰WT1基因表达的小鼠足细胞Wnt/β-catenin信号通路的变化及其对足细胞活力和nephrin表达的影响。方法:采用RPMI-1640培养基在33℃条件下传代培养足细胞,在37℃条件下诱导足细胞分化。应用WT1基因的siRNA转染分化成熟的小鼠足细胞,用MTT法测定细胞活力,用实时qRT-PCR和Western blotting技术分别检测mRNA和蛋白的表达。结果:RNA干扰WT1可诱导小鼠足细胞Wnt1mRNA和蛋白表达上调,p-β-catenin水平降低及足细胞活力下降,伴有足细胞nephrin mRNA和蛋白的表达明显下调。结论:沉默WT1基因的表达可诱导足细胞活力下降和nephrin表达下调,可能与Wnt/β-catenin信号通路的激活有关。更多还原

【Abstract】 AIM: To study the effect of WT1 silencing by small interfering RNA(siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes.METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃.The podocytes were transfected with WT1 siRNA.The cell vitality was detected by MTT assay.The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting.RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels,inhibited the phosphorylation of β-catenin,and reduced the cell vitality.Meanwhile,the expression of nephrin at mRNA and protein levels was decreased.CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.更多还原