NADPH氧化酶在TGF-β_1诱导大鼠小管上皮细胞表达炎症因子中的作用  

Role of NADPH Oxidade in TGF-β_1-Induced Inflammatory Factors Expression in Rat Renal Tubular Epithelial Cells

作　　者：张海燕[1] 姜宗培[1] 常洁[1] 李晓艳[1] 朱恒梅[1] 余学清[1] 

机构地区：[1]中山大学附属第一医院肾内科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2008年第3期252-257,共6页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：教育部留学回国人员科研启动基金(教外司[2004]176);广东省科技计划项目(2004B30701002);广东省中医药局项目(103053)

摘　　要：【目的】观察转化生长因子β1(TGF-β1)对大鼠肾小管上皮细胞单核细胞趋化因子-1(MCP-1)及白细胞介素-6(IL-6)表达的影响,并探讨NADPH氧化酶在其中的作用。【方法】以大鼠肾小管上皮细胞株(NRK-52E)为研究对象,采用TGF-β1(10ng/mL)刺激0h,2h,4h,8h,12h,24h分别收取RNA;刺激0h,12h,24h,48h,72h分别收取细胞上清液;部分实验中培养的细胞在刺激前用NADPH氧化酶抑制剂DPI(0.1,1,5,10μmol/L)预处理1h,然后含10ng的TGF-β1无血清DMEM/F12培养液分别培养12h(收集RNA)和24h(收集细胞上清液)。所有实验重复3次。细胞内活性氧(ROS)的产生采用激光共聚焦显微镜观察。NADPH氧化酶p22phox、gp91phox、p47phox和p67phox亚单位以及MCP-1和IL-6mRNA的表达采用RT-PCR检测。细胞上清液中MCP-1的含量采用ELISA检测。【结果】TGF-β1可显著上调NADPH氧化酶p67phox亚单位mRNA的表达,8h为对照的2.43倍(P<0.01),24h达3.59倍(P<0.01)。但对p22phox、gp91phox和p47phox亚单位mRNA表达无显著影响。TGF-β1可显著增加细胞内ROS产生,DPI预处理后ROS产生较TGF-β1刺激组降低了43.69%(P<0.05)。TGF-β1可显著上调MCP-1和IL-6mRNA及蛋白的表达。DPI预处理可明显逆转TGF-β1诱导的MCP-1和IL-6的上调表达。【结论】TGF-β1可促使细胞ROS产生增加。TGF-β1可能通过NADPH氧化酶依赖的ROS介导了大鼠肾小管上皮细胞MCP-1及IL-6的上调表达。[Objective] To investigate the role of NADPH oxidase in TGF-β1-induced monocyte chemoattractant protein-1 （MCP-1）and interleukin-6 （IL-6） in rat renal tubular epithelial cells. [Methods] Growth arrested and synchronized rat epithelial cells （NRK-52E） were stimulated by 10 ng/mL TGF-β1 for 0 h, 2 h, 4 h, 8 h, 12 h, 24 h for RNA and 0 h, 12 h, 24 h, 48 h, 72 h for protein. In some experiments, cells were pretreated with different concentration of DPI （0.1, 1, 5, 10 μmol/L）, an inhibitor of NADPH oxidase, for lh, and then were stimulated by 10 ng/mL TGF-β1 for 12 h （MCP-1 and IL-6 mRNA expression） and 24 h （MCP-1 protein secreation）, respectively. Intracellular ROS generation was visualized using H2DCF-DA by confocal. The mRNA expression of NADPH oxidase subunits, MCP-1 and IL-6 in cultured NRK-52E cells were measured by semi-quantitive reverse transcriptase polymerase chain reaction （RT-PCR）. The MCP-1 protein levels in the medium were assessed by ELISA assay. [Results] 10 ng/mL TGF-β1 upregulated p67phox mRNA by 2.43-fold at 8 h （P 〈 0.01） and 3.59-fold at 24 h （P〈 0.01） when compared with control,but no effect on p22phox, gp91phox, and p47phox mRNA expression. TGF-β1 can significantly increase intracellular ROS. DPI can decrease the generation of ROS induced by TGF-β1 by 43.69% （P 〈 0.05）. TGF-β1 significantly increased the MCP-1 mRNA and protein and IL-6 mRNA expression, DPI effectively reversed TGF-β1-induced MCP-1 and IL-6 expression in rat renal tubular epithelial cells. [Conclusion] TGF-β1 can significantly increase intracellular ROS in rat renal tubular epithelial cells. TGF-β1 stimulated the MCP-1 and IL-6 expression may be mediated by NADPH oxidase-derived ROS.

关 键 词：转化生长因子β NAD(P)H氧化酶 活性氧 单核细胞趋化蛋白-1 白细胞介素-6 

分 类 号：R363[医药卫生—病理学]