环氧合酶-2基因敲低对足细胞凋亡及nephrin蛋白表达的影响
The effect of COX-2 gene knockdown on podocyte apoptosis and nephrin protein expression
孙怀鑫 1孔维信 1汪年松 2张林 1丁露露 1郝传明 3陈靖3
作者信息
- 1. 上海交通大学医学院苏州九龙医院肾内科,江苏苏州215021
- 2. 上海市第六人民医院肾内科,上海200040
- 3. 复旦大学附属华山医院肾内科,上海200032
- 折叠
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-X1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,15 μl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,10μlCOX-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-X1蛋白下调参与了足细胞的凋亡.
Abstract
Objective To investigate the effect of COX-2 gene knockdown on podocyte apoptosis and nephrin protein expression. Methods The conditionally immortalized murine podocytes were cultured and randomly divided into four groups: normal control group, podocytes treated with 5μ1 of small interfere RNA (siRNA),podocytes treated with 10 μ1of siRNA, podocytes treated with 15 μ1 of siRNA. In differentiated podocytes, the expression of COX-2 was knockdown by siRNA. The mRNA and protein expression of COX-2 were detecteded by semi-quantitative RT-PCR and western blotting respectively. The level of podocyte apoptosis was measured by flow cytometry. And then the 10 μ1 of siRNA group was selected for cells were divided into further study, normal control group, podocytes treated with 10μ1 of siRNA and podocytes treated with negative siRNA were set to study. The protein expression levels of nephrin and BAX as well as BCL-X1 were detected by western blotting. Results The expressions of COX-2 mRNA and protein were decreased with the concentration of siRNA increasing. To the contrary, the podocytes apoptosis were increased. The apoptosis of podocytes treated with 15μ1 of siRNA significantly increased. And the protein expression level of nephrin in 10 μ1 of siRNA group was significantly lower than that of in negative siRNA group. The protein expression level of Bax in 10μ1 of siRNA group was significantly higher than in that of in negative siRNA group, However the protein expression level of Bcl-xl was lower than that of in negative siRNA group. Conclusion The COX-2 gene knockdown induces podocytes apopto-sis. With the concentration of siRNA increasing, the apoptosis rates of podocytes were gradually increased. At the concentration of 10μ1 of siRNA, the protein expression level of nephrin in podocyte was downregulated. The upregulation of Bax and the downregulation of Bcl-xl may contribute to podocyte apoptosis.
关键词
足细胞/细胞凋亡/基因敲低Key words
podocyte/apoptosis/knockdown引用本文复制引用
基金项目
苏州市科技计划项目(SS0716)
苏州工业园区孵化项目()
出版年
2012
NETL
NSTL
维普
万方数据