香烟烟雾提取物诱导呼吸道上皮细胞间质性转化的实验研究  

Cigarette smoke extrac induced epithelial-mesenchymal transition in respiratory epithelial cells

作　　者：刘振峰[1] 刘建英[1] 刘代顺[1] 周国旗[1] 陈乾[2] 王海霞 

机构地区：[1]贵州省遵义市第一人民医院呼吸科,563003 [2]贵州省遵义市第一人民医院神经内科,563003 [3]江苏省苏州市科技城医院呼吸科,215000

出　　处：《重庆医学》2017年第3期318-321,共4页Chongqing medicine

基　　金：贵州省科学技术基金项目(黔科合J字[2013]2301)

摘　　要：目的探讨香烟烟雾提取物(CSE)在呼吸道上皮细胞间质性转化(EMT)中的作用,为进一步研究香烟烟雾提取物在慢性阻塞性肺疾病(COPD)气道重构发生机制提供新的线索。方法用不同浓度(0%、0.5%、1.0%、2.5%、5.0%,其中浓度0作为对照组)的CSE刺激呼吸道上皮细胞BEAS-2B72h,逆转录PCR检测E钙粘蛋白(E-cad)、N钙粘蛋白(N-cad)mRNA表达情况,酶联免疫吸附试验(ELISA)检测细胞培养上清液中TGF-β1浓度;用2.5%CSE刺激BEAS-2B细胞不同时长(0、12、24、48h,其中时间0h作为对照),倒置显微镜下观察细胞形态变化,逆转录PCR检测E-cad、N-cad、补体C3mRNA表达情况,ELISA法检测细胞培养上清液中TGF-β1、过敏毒素C3a浓度。结果在不同浓度的CSE刺激BEAS-2B细胞72h后,CSE各浓度刺激组较对照组E-cad mRNA表达下调,N-cad mRNA表达增加,且呈浓度依赖性(P<0.05);ELISA法检测细胞培养上清液中TGF-β1水平,CSE各浓度刺激组较对照组增加(P<0.05);2.5%CSE在不同时间点作用BEAS-2B细胞,刺激组细胞呈梭形改变,CSE各时间段刺激组较对照组E-cad mRNA表达减少,补体C3、N-cad mRNA表达增加,且呈时间依赖性(P<0.05),CSE各时间段刺激组细胞培养上清液中TGF-β1、C3a水平较对照组增加(P<0.05)。结论 CSE可诱导正常的呼吸道上皮细胞BEAS-2B发生EMT,补体C3激活可能参与此过程。Objective To investigate the epithelial-mesenchymal transition in respiratory epithelial cells stimulated by cigarette smoke extract in vitro for the new clue of the airway remodeling of COPD due to cigarette smoke.Methods The messenger ribonucleic acid levels of E-cadherin,N-cadherin were measured by RT-PCR combined with agarose gel electrophoresis after the treatment of different concentrations cigarette smoking extract（0%,0.5%,1.0%,2.5%,5.0%）for 72 hours in respiratory epithelial cells.The concentration of TGF-β1was quantified by enzyme linked immunosorbent assay in the supermatant.After the treatment of 2.5% cigarette smoking extract in respiratory epithelial cells,the morphological changes measured by inversion microscope,the messenger ribonucleic acid levels of E-cadherin,N-cadherin and complement component C3 by RT-PCR combined with agarose gel electrophoresis and the protein concentration of TGF-β1and complement component C3 ain the supermatant by enzyme linked immunosorbent assay were conducted at different times（0,12,24,48h）.Results The stimulation of different concentrations CSE for72 hours in BEAS-2Bcells decreased the level of E-cadherin mRNA expression accompanied by rising expression of the messenger ribonucleic acid level of N-cadherin compared with the control group in a concentration dependent manner,Simultaneously,after different stimulations there was a significant increase in TGF-β1protein level in the supermatant（P〈0.05）.After the treatment of2.5% cigarette smoking extract in respiratory epithelial cells at different times,BEAS-2Bcells acquired an elongated,shuttle-like shape in morphology,besides which there was an inverse change that the mRNA expression of E-cadherin was reduced compared with the control group as a significant increase in a time dependent manner in the mRNA expression of N-cadherin and complement component C3（P〈0.05）,as well as the protein level of TGF-β1and complement component C3 ain the supermatant were elevated after BEAS-2Bcells treated with 2.

关 键 词：肺疾病 慢性阻塞性 香烟烟雾提取物 呼吸道上皮细胞 上皮间质转化 过敏毒素C3a 

分 类 号：R563.9[医药卫生—呼吸系统]