环氧合酶-2基因敲低对足细胞凋亡及podocin蛋白表达的影响  

Effect of COX-2 gene knockdown on podocyte apoptosis and podocin protein expression

作　　者：张林[1] 孔维信[1] 孙怀鑫[1] 丁露露[1] 郝传明[2] 陈靖[2] 

机构地区：[1]上海交通大学医学院苏州九龙医院,江苏苏州215021 [2]复旦大学附属华山医院

出　　处：《山东医药》2012年第33期18-21,共4页Shandong Medical Journal

基　　金：苏州市科技计划项目(SS0716)

摘　　要：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podocin蛋白表达的影响,以进一步探讨足细胞凋亡的分子机制。方法将条件永生性小鼠足细胞株随机分为阴性转染组、5μL siRNA组、10μL siRNA组、15μL siRNA组。经诱导分化成熟,在siRNA干扰48 h后用半定量RT-PCR和Western bolt分别检测足细胞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。将10μL siRNA组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三个亚组,用Western bolt检测podocin蛋白、BAX蛋白及BCL-X1蛋白的表达水平。结果与阴性转染组比较,随着干扰浓度的增加,COX-2 mRNA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。足细胞COX-2基因敲低组podocin蛋白表达显著低于足细胞阴性转染对照组(P<0.05)。与足细胞阴性转染对照组相比,足细胞COX-2基因敲低组BAX蛋白表达显著上调,而BCL-X1蛋白表达稍有下调。结论敲低COX-2基因表达可以导致足细胞凋亡,且随着siRNA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podocin蛋白的表达下调。BAX蛋白上调及BCL-X1蛋白下调参与了足细胞的凋亡。Objective To investigate the effect of COX-2 gene knockdown on podocyte apoptosis and podocin protein expression and to explore the molecular mechanism of podoeyte apoptosis. Methods The conditionally immortalized murine podocytes were randomly divided into four groups ： normal control group, podocytes treated with 5 μL of small interfere RNA （siRNA）, podocytes treated with 10 μ1 of siRNA, podocytes treated with 15 μL of siRNA. In differentiated podocytes, the expression of COX-2 was knockdown by siRNA. The mRNA and protein expressions of COX-2 were detected by semi-quantitative RT-PCR and western blot respectively. The level of podocyte apoptosis was measured by flow cytometry. And then the 10 μL of siRNA group was selected to further study. Normal control group, podocytes treated with 10 μL of siRNA and podocytes treated with negative siRNA were set to study. The protein expression levels of podocin and BAX as well as BCL-X1 were detected by western blot. Results The expressions of COX-2 mRNA and protein were decreased over concentration of siRNA. To the contrary, the podocytes apoptosis were increased. The apoptosis of podocytes treated with 15 μL of siRNA significantly increased. And the protein expression level of nephrin in 10 μL of siRNA group was significantly lower than that in negative siRNA group（P 〈0.05 ）. The protein expression level of BAX in 10 μL of siRNA group was significantly higher than that in negative siPtNA group, however the protein expression level of BCL-X1 was lower than that in negative siRNA group. Conclusion The COX-2 gene knockdown induces podocytes apoptosis in concentration-de- pendent mode. At the concentration of 10 μL of siRNA, the protein expression level of podocin in podocyte was downregnlated. The upregnlation of BAX and the downregulation of BCL-X1 may contribute to podocyte apoptosis.

关 键 词：足细胞 细胞凋亡 基因敲低 

分 类 号：Q343[生物学—遗传学]