NADPH氧化酶在TGF-β1诱导的大鼠腹膜间皮细胞转分化中的作用  

Effects of NADPH oxidase on TGF-β1-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells

作　　者：刘小贤[1] 周红娟[1] 汪卫[1] 蔡龙[1] 于健宁[1] 马纪林[1] 张雯[1] 

机构地区：[1]浙江省中西医结合医院肾内科,杭州310003

出　　处：《浙江医学》2012年第19期1555-1558,1565,共5页Zhejiang Medical Journal

基　　金：基金项目：浙江省自然科学基金资助项目（Y2101241）

摘　　要：目的探讨NADPH氧化酶在TGF—β1诱导的大鼠腹膜间皮细胞（PMCs）转分化中的作用。方法体外培养SD大鼠原代PMCs．静止24h后，随机分为对照组（A组）、DPI（NADPH氧化酶抑制剂，10μmol／L）组（B组），TGF—β1（10μg／L）组（C组），TGF—β1＋DPI（10μmol／L）组（D组，DPI预处理1h）。观察NADPH氧化酶亚单位p67phox、活性氧（ROS）、α-平滑肌肌动蛋白（α—SMA），E-钙粘素（E—cadherin）和Ⅰ型胶原（Collagen Ⅰ）的表达。用荧光染料（DCF）及激光共聚焦显微镜检测细胞内ROS。分别用RT—PCR方法和Western印迹检测p67phox、E—cadherin、α-SMA和Collagen ⅠmRNA和蛋白的表达情况。结果TGF—β1显著增加了ROS的产生，刺激24h达到高峰；DPI可抑制TGF—β1诱导的ROS的产生；DPI可抑制TGF—β1诱导的NADPH氧化酶亚单位p67phoxmRNA和蛋白的表达上调，与单纯TGF—β1刺激组比较有统计学差异（P〈0.05）；与此同时DPI可显著抑制TGF—β1诱导的E—cadherin表达下调和α-SMA、CollagenⅠ表达的上调。结论依赖NADPH氧化酶的ROS介导了TGF—β1诱导的大鼠PMCs转分化，NADPH氧化酶可能是腹膜纤维化防治的潜在靶点。Objective TO investigate the effect of NADPH oxidase on TGF- β1-induced epithelial-mesenchymal transition （EMT） in rat peritoneal mesothelial cells （PMCs）. Methods Primary rat peritoneal mesothelial cells were cultured into the second generation in vitro. After synchronization for 24h, the cells were randomly assigned to Groups A （no treatment）, B （treated with 10 μ mol/L NADPH oxidase inhibitor diphenyleneiodonium, DPI）, C （treated with 10μ g/L TGF- β 1） and D （pretreated with 10 μ mol/L DPI for lh before 10 μg/L TGF-β 1 stimulation）. The DCF-sensitive cellular reactive oxygen species （ROS） was measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression of NADPH oxidase subunit p67phox, E-cadherin, α -smooth muscle actin （ α-SMA） and type Ⅰcollagen （Col Ⅰ ）. Expressions of E-cadherin, -SMA, Col I and p67phox proteins were examined by Western blot analysis. Results TGF-β 1 significantly induced the production of intracellular ROS compared with control （P〈0.05）; ROS expression increased more markedly after stimulation for 24h. DPI inhibited TGF- β 1 induced ROS generation by 46.34% （P〈0.05）. TGF- β1 significantly stimulated NADPH oxidase subunit p67phox mRNA and protein expression in RPMCs, and DPI inhibited TGF- β 1-induced up-regulation of p67phox mRNA and protein by 39.3% and 47.8% （P〈0.05）, respectively. TGF- β 1 significantly upregulated mRNA and protein expression of α-SMA and Col Ⅰ , and downregulated mRNA and protein expression of E-cadherin in rat PMCs. This effect was significantly suppressed by DPI. Conclusion NADPH oxidase-dependent formation of ROS mediates TGF-β 1 dependent EMT in rat peritoneal mesothelial cells. NADPH oxidase may represent a potential therapeutic target in the management of peritoneal fibrosis.

关 键 词：NADPH氧化酶 腹膜间皮细胞 转分化 TGF—β 

分 类 号：R495.5[医药卫生—康复医学]