【作者】 杨毅；

【导师】 凃晋文；

【作者基本信息】 湖北中医学院 ， 中医内科， 2009， 博士

【摘要】 研究背景:肺纤维化(pulmonary fibrosis,PF)是以运动性呼吸困难进行性加重,以喘息、气短、干咳、喘憋为临床表现,以限制性通气功能障碍、低氧血症、慢性进行性弥漫性肺间质纤维化为特点的肺间质性疾病。肺纤维化既是多种肺疾病、多种系统性疾病的共同结局,又有无原发疾病的特发性肺间质纤维化。近年来,随着细胞和分子生物学等生命科学理论研究,多种研究手段的应用,从器官、组织、细胞、分子的不同层次,对肺纤维化的发生、诊断、防治的研究不断深入,取得极大的进展。研究表明,肺纤维化是由早期组织损伤、炎症变化发展而来,而肺部受感染或损伤后,肺泡上皮细胞和是最早受到损伤的靶细胞。由于肺泡表面约93-97%的肺泡Ⅰ型上皮细胞为终末细胞,不能进行分裂增殖,故具有增殖分化能力的肺泡Ⅱ型上皮细胞(Alveolar typeⅡcell,ATⅡ)是参与组织损伤修复的主要细胞,所以,最初肺损伤后发生于组织修复时的肺泡Ⅱ型上皮细胞向间质细胞转化(epithelial to mesenchymaltransition,EMT)在肺纤维化发生发展过程中发挥着极其重要的作用。近年来随着对肺纤维化的发病机理的深入研究,无论中、西医均在治疗上取得相当进展。导师在长期的临床实践中,依照经验创立苈桃抗纤方,对肺间质纤维化患者进行雾化治疗,取得了一定的疗效。为了提高它的可信度和可重复性,为临床实践提供更好生命科学理论基础,在本实验中,我们主要研究苈桃抗纤方对肺泡Ⅱ型上皮细胞向问质细胞转化、EMT转化率以及对肺泡Ⅱ型上皮细胞转化过程中相关细胞转导通路的影响,在细胞及分子水平上探讨苈桃抗纤方抗肺纤维化的生物学基础。目的:由于肺泡Ⅱ型上皮细胞向间质细胞转化(EMT)是发生肺纤维化的重要因素,故在本次实验中,我们应用转化生长因子(transforminggrowth factor TGF-β1)诱导人肺上皮细胞系(A549)EMT发生,观察TGF-β1对A549增殖及形态变化的影响,研究苈桃抗纤方雾滴干预后对肺泡Ⅱ型上皮细胞向间质细胞转化的影响,从基于雾化中药制剂影响EMT发生的角度,初步探讨苈桃抗纤方防治肺纤维化的生物学基础。方法:使用不同浓度苈桃抗纤方体外干预A549细胞。免疫组化法检测细胞内TGF-β1的相对含量;通过TGF-β1体外诱导细胞EMT的发生,ELISA法检测细胞角蛋白(cytokeratin)及波形蛋白(Vimentin)表达,计算细胞EMT转化率;Real-time PCR法检测苈桃抗纤方对细胞内Ⅰ型胶原及Ⅲ型胶原mRNA表达的影响;Western Blot检测细胞磷酸化(phosphorylation)Erk1/2的表达。结果:不同浓度苈桃抗纤方体外作用A549细胞48h后,胞内TGF-β1的定量表达,相较正常对照组明显减少(40.35±1.65,37.84±0.95,37.45±0.78 VS 45.13±2.01),(P<0.01)具有显著差异性。TGF-β1体外诱导A549细胞EMT发生后,ELISA检测细胞cytokeratin和vimentin表达显示,相较正常对照组,vimentin阳性表达明显增强,而cytokeratin阳性表达相对减弱;RT-PCR检测表明,A549细胞内Ⅰ型胶原及Ⅲ型胶原mRNA表达显著增强。经不同浓度苈桃抗纤方体外作用诱导EMT成功的A549细胞48h后,与模型组相比,vimentin阳性表达出现不同程度的减弱,而cytokeratin阳性表达则出现不同程度增强;Ⅰ型胶原及Ⅲ型胶原mRNA表达均出现不同程度下调;phosphorylation Erk1/2的表达减弱。结论:苈桃抗纤方可以降低TGF-β1诱导的A549细胞EMT转化率,阻断肺泡Ⅱ型上皮细胞向间质细胞转化,减少肺成纤维细胞的产生,从而抑制肺纤维化的发生,达到抗肺纤维化的作用。其可能机制是通过降低细胞内转化生长因子(transforming growth factor TGF-β1)的释放,干预Ⅰ型及Ⅲ型胶原mRNA表达,影响细胞磷酸化Erk1/2的表达。更多还原

【Abstract】 Objective:Pulmonary fibrosis results from infection,drugs, chemicals,organic or inorganic dust,and other autoimmune diseases,that stimulate alveolar damage.It is a leading cause of Chronic obstructive pulmonary disease、Idiopathic pulmonary fibrosis and other serious lung diseases.As the alveolar epithelial typeⅡcells to mesenchymal cell transformation(EMT) is an important factors of pulmonary fibrosis,so we adopted A549 cells in this experiment and used transforming growth factor induced EMT to occur in A549 cells to study the effect of Li Tao Kang Xian Fang droplet in EMT.Real-time PCR,Western-blot and ELISA was introduced to define the biology basic of Li Tao Kang Xian Fang droplet playing a anti- fibrosis role by controling EMT.Methods:Different concentrations of Li Tao Kang Xian Fang was used to intervene A549 cells in vitro.ELISA were used to show the retative contents of the TGF-β1 in A549 cells.We used TGF-β1 to induce the occurrence of EMT.The collagenⅠand collagenⅢmRNA level was monitored by real-time PCR,the protein level of phosphorylation Erk1/2 were examined by western blot analysis. Cell apoptosis and cell cycle distribution were determined by flow cytometry.The protein level of phosphorylation Erk1/2 were examined by western blot analysis.Results:Our data showed that contents of the TGF-β1 ruduced after 24h intervention of different concentrations of Li Tao Kang Xian Fang.In TGF-β1-induced cells,vimentin espression have a sharp increase but cytokeratina espression have a relative reduction.The level of collagenⅠandⅢshow Significantly higher.By intervening of different concentrations of Li Tao Kang Xian Fang,vimentin espression was significantly lower,yet cytokeratina espression was significantly higher than that in the model group.Meantime,It bring a obvious reduction in espression of collagenⅠ、Ⅲand phosphorylation Erk1/2.Conclusion:Li Tao Kang Xian Fang can drop the EMT rates of TGF-β1 -induced A549 cells,thereby ruduce ACEⅡcells to epithelial cells.It lead to a reduction of lung fibroblast production,to inhibit the occurrence of pulmonary fibrosis,so as to achieve an anti- fibrosis role.The possible mechanism maybe by reducing the transforming growth factor(TGF-β) releasion,interveningⅠandⅢcollagen mRNA expression,and affecting cell phosphorylation Erk1/ 2 expression.更多还原