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目的观察足细胞环氧合酶-2(COX-2)基因敲低对足细胞凋亡及nephrin蛋白表达的影响。方法 (1)以条件永生性小鼠足细胞株为研究对象:分为正常对照组、5μl的siRNA组、10μl的siRNA组、15μl的siRNA组。经诱导分化成熟,在干扰48 h后用半定量RT-PCR和Western bolt分别检测COX-2 mRNA及蛋白表达水平,用流式细胞仪检测足细胞凋亡水平。(2)以10μl的siRNA组作为研究对象,分别设立足细胞正常对照组、足细胞COX-2基因敲低组和足细胞阴性转染对照组,用Western bolt检测nephrin蛋白、BAX蛋白及BCL-Xl蛋白的表达水平。结果 (1)与正常对照组比较,随着干扰浓度的增加,COX-2mRNA及蛋白表达逐渐减少,而足细胞的凋亡率逐渐升高,15μl的siRNA组凋亡率明显增高。(2)1μl COX-2 siRNA转染组nephrin蛋白表达显著低于阴性转染组(P<0.05)。(3)与阴性转染组相比,10μl COX-2 siRNA转染组Bax蛋白表达显著上调,而Bcl-xl蛋白表达稍有下调。结论敲低COX-2的基因表达可以导致足细胞凋亡,并随着干扰浓度的增加,足细胞凋亡率逐渐增加;在干扰浓度为10μl时,足细胞nephrin蛋白的表达下调;BAX蛋白上调及BCL-Xl蛋白下调参与了足细胞的凋亡。
Objective To observe the effect of podocyte cyclooxygenase-2 (COX-2) gene knockdown on podocyte apoptosis and nephrin protein expression. Methods (1) The conditional immortalized mouse podocyte strain was divided into normal control group, 5μl siRNA group, 10μl siRNA group and 15μl siRNA group. After induction of differentiation and maturation, the expression of COX-2 mRNA and protein were detected by semi-quantitative RT-PCR and Western bolt respectively 48 h after interference, and the apoptosis of podocytes was detected by flow cytometry. (2) 10μl siRNA group was used as the research object, and the normal control group, the COX-2 knockdown group and the podocyte negative control group were established respectively. The nephrin protein, BAX protein and BCL- Xl protein expression level. Results (1) Compared with the normal control group, the expression of COX-2 mRNA and protein decreased with the increase of the interference concentration, while the apoptosis rate of podocytes gradually increased. The apoptosis rate of 15μl siRNA group was significantly increased. (2) The nephrin protein expression in 1μl COX-2 siRNA transfected group was significantly lower than that in negative transfected group (P <0.05). (3) Bax protein expression was significantly up-regulated and Bcl-xl protein expression was slightly down-regulated in 10μl COX-2 siRNA transfection compared with negative transfection group. Conclusion The knockdown of COX-2 gene expression can lead to podocyte apoptosis, and the podocyte apoptosis rate gradually increases with the increase of the interference concentration. The podocyte nephrin protein expression is down-regulated and BAX protein is up-regulated at the interference concentration of 10μl And BCL-Xl protein is involved in the podocyte apoptosis.