β－连环素通路对转化生长因子β1致肺纤维化的调控作  

Regulation effect of β-catenin pathway on TGF-β1 induced pulmonary pro-fibrosis

作　　者：田新瑞[1] 田新利[2] 王慧芳[3] 常琴[1] 霍如婕 殷得莉 郑国平[4] 

机构地区：[1]山西医科大学第二医院呼吸与危重症医学科,太原030001 [2]北京军区总医院心肺中心 [3]山西大同市第五人民医院呼吸内科 [4]山西医科大学生物化学与分子生物学系

出　　处：《中华医学杂志》2016年第24期1929-1933,共5页National Medical Journal of China

基　　金：山西省自然科学基金（2013011055-1）;山西省归国留学人员科研资助项目（2011-104）：山西省自然科学基金（2013011055-1）;山西省归国留学人员科研资助项目（2011-104）

摘　　要：目的探讨β－连环素通路对转化生长因子β1（TGF－β1）致肺纤维化的调控作用。方法体外培养的大鼠肺泡Ⅱ型上皮细胞（RLE－6TN细胞）分为四组：A1.对照组；B1.加5 μg/L TGF－β1的TGF－β1组；C1.转染真核表达载体pcDNA3.0（pcDNA）后加5 μg/L TGF－β1的pcDNA＋TGF－β1组；D1.转染β－连环素敲除载体[F－（β－TrCP）－Ecad]后加5 μg/L TGF－β1的F－（β－TrCP）－Ecad＋TGF－β1组。24 h后，显微镜下观察各组细胞形态，Western印迹法检测各组细胞上皮钙黏素、α－平滑肌肌动蛋白（α－SMA）和纤维连接蛋白（Fn）表达；实时荧光定量－聚合酶链反应（RT－PCR）检测各组细胞培养上清液中促纤维化转录产物Snail mRNA的表达。体外培养的大鼠肺泡巨噬细胞（CRL－2192细胞）分为五组：A2.对照组；B2.加20 μg/L干扰素γ的干扰素γ组；C2.加10 μg/L TGF－β1和20 μg/L干扰素γ的TGF－β1＋干扰素γ组；D2.转染F－（β－TrCP）－Ecad后加入C2组相同试剂的F－（β－TrCP）－Ecad＋TGF－β1＋干扰素γ组；E2.转染野生型β－连环素表达载体（WTβ－连环素）后加入C2组相同试剂的WTβ－连环素＋TGF－β1＋干扰素γ组。24 h后，Western印迹法检测A2、B2、C2组中β－连环素蛋白的表达；RT－PCR检测各组细胞培养上清液诱导型一氧化氮合酶（iNOS）mRNA的表达。结果B1、C1组RLE－6TN细胞形态变为梭形，D1组细胞形态基本呈铺路石样；B1、C1组RLE－6TN细胞纤维细胞特征蛋白α－SMA、Fn蛋白及下游促纤维化转录产物Snail mRNA表达均显著高于A1组，而上皮细胞特征蛋白上皮钙黏素蛋白表达显著低于A1组；D1组细胞α－SMA、Fn蛋白、Snail mRNA表达均显著低于C1组（0.352±0.076比0.937±0.303、0.319±0.072比0.903±0.211、3.675±0.642比9.708±2.031），而上皮钙黏素蛋白表达显著高于C1组（1.482±0.227比0.604±0.121）（均P〈0.05）。CRL－2192细胞中，β－连�ObjectiveTo investigate the regulation effect of β-catenin pathway on transforming growth factor beta1 （TGF-β1） induced pulmonary pro-fibrosis.MethodsThe rat alveolar typeⅡ cells （RLE-6TN） were divided into four groups： A1.control group; B1.TGF-β1 group was treated with 5 μg/L TGF-β1; C1.pcDNA＋ TGF-β1 group was transiently transfected with eukaryotic expression vector pcDNA3.0 （pcDNA） and followed by TGF-β1 treatment （5 μg/L）; D1.F-（β-TrCP）-Ecad＋ TGF-β1 group was transiently transfected with β-catenin protein knockout vector [F-（β-TrCP）-Ecad] and followed by TGF-β1 treatment （5 μg/L）. After 24 hours, cells were observed under the inverted phase contrast microscope, then the expressions of E-cadherin, α-smooth muscle actin （α-SMA） and fibronectin （Fn） in each group were measured by Western blot and the mRNA levels of Snail which was the downstream profibrotic transcription production in cell culture supernatants of each group were detected by real-time fluorescence quantification-polymerase chain reaction （RT-PCR） .The rat alveolar macrophages （CRL-2192） were divided into five groups： A2.control group; B2.Interferon gamma （IFN-γ） group was treated by 20 μg/L IFN-γ; C2.TGF-β1＋ IFN-γ group was treated by 20 μg/L IFN-γ with 10 μg/L TGF-β1; D2.F-（β-TrCP）-Ecad＋ TGF-β1＋ IFN-γ group was transfected with F-（β-TrCP）-Ecad and other dispose was the same as group C2; E2.WTβ-catenin＋ TGF-β1＋ IFN-γ group was transfected with WTβ-catenin and other dispose was the same as group C2.After 24 hours, protein levels of β-catenin in group A2, B2, C2 were determined by Western blot.Inducible nitric oxide synthase （iNOS） mRNA levels of each group were detected by RT-PCR.ResultsThe RLE-6TN cells of group B1, C1 showed a change in morphology to spindle-shaped cells, the cells of group D1 maintained a cobblestone morphology. Protein expressions of the fibroblast markers α-SMA and Fn, and mRNA expressions of the downstream profi

关 键 词：肺纤维化 Β连环素 转化生长因子Β1 上皮细胞-间质转化 巨噬细胞 

分 类 号：R563[医药卫生—呼吸系统]