【作者】 李军利；

【导师】 陈平；

【作者基本信息】 中南大学 ， 呼吸内科， 2009， 博士

【摘要】 研究背景:慢性阻塞性肺疾病（COPD）是严重影响人类健康的常见多发病,患病人数多,死亡率高,社会经济负担重,是全球性公共卫生问题。该病发病机制未完全明了,且无理想的治疗方法。烟雾暴露是COPD的重要致病因素。除慢性气道和肺部炎症、蛋白酶/抗蛋白酶失衡以及氧化/抗氧化失衡等学说外,肺间隔细胞凋亡在COPD发生发展中的作用日益受到重视,且与上述学说有千丝万缕的联系。肺间隔细胞凋亡的累积效应将导致肺气肿的发生和呼吸功能的降低,导致呼吸衰竭的发生。因此,干预肺间隔细胞凋亡是目前COPD防治研究中的一项重要课题。转录因子活化蛋白-2（transcription factoractivator protein-2,AP-2）家族是一组发育调节,维甲酸诱导的基因DNA结合蛋白,AP-2α（transcription factor activator protein-2α,AP-2α）是最早被鉴定和研究的AP-2家族成员,参与了许多基因的表达如VEGF、P⁵³、p^21WAF/CIP、Bcl-2、锰过氧化物岐化酶（Manganese superoxidedismutase,MnSOD）等;参与了脊椎动物细胞黏附、生长、凋亡、组织分化、胚胎发育、肿瘤发生等病理生理过程。其中大部分研究集中在AP-2α对增殖凋亡的调控上。依据细胞类型及实验条件的不同,AP-2α分别发挥促凋亡或促增殖的作用。但是,在烟雾暴露所致的COPD大鼠肺组织及香烟提取物（CSE）刺激的人脐静脉内皮细胞株ECV304中AP-2α是否表达,是否对烟雾暴露引起的细胞凋亡及Caspase-3的活化有影响,其机制是否与其激活结构域有关等等,尚无相关报道。目的:研究熏烟所致COPD大鼠肺组织中肺间隔细胞的凋亡情况及AP-2α的表达水平;进一步通过细胞实验研究AP-2α对CSE诱导的ECV304凋亡的影响及机制。方法:采用被动吸烟80天的方法复制大鼠COPD模型,采用TUNEL法检测肺组织细胞凋亡、Western blot法检测肺组织中活化的Caspase-3、免疫组织化学及Western blot法检测胞核中AP-2α蛋白的表达;通过MTT、Hoechst染核、Western blot等方法研究CSE对ECV304增殖、凋亡、Caspase-3和AP-2α表达的影响;通过腺病毒介导的转染、Hoechst染核、Western blot等方法研究过表达AP-2α对CSE诱导的ECV304凋亡、Caspase-3活化的影响。采用质粒介导的转染、Hoechst染核、Western blot等方法研究过表达AP-2α的突变体AP-2Δ（只具有结合结构域,而没有转录激活结构域）对CSE诱导的ECV304凋亡、Caspase-3活化的影响。数据以均数±标准差（x±s）表示,用Bartlett法进行方差齐性检验,两组间比较用t检验,多组间比较用单因素方差分析,多个样本均数的多重比较用LSD-t检验。多个样本率的比较用X²检验。结果:研究发现,烟雾暴露80天成功复制了COPD大鼠模型,表现为炎症细胞浸润,肺泡腔扩大,FEV_0.3/FVC降低,肺间隔细胞凋亡增多,Caspase-3活化增加;同时AP-2α蛋白表达增加。在CSE刺激的ECV304细胞中,细胞凋亡增多,AP-2α表达增加;AP-2α的过表达可抑制CSE诱导的ECV304细胞凋亡,表现为细胞凋亡率降低和Caspase-3的活性片段（17KD）减少。AP-2Δ瞬时转染ECV304细胞并使其过表达后对基础及CSE诱导的细胞凋亡、Caspase-3的活化没有明显影响。结论:AP-2α在烟雾暴露法复制的COPD大鼠肺组织及CSE刺激的ECV304细胞中表达增加。在CSE刺激的ECV304细胞中,AP-2α抑制CSE诱导的Caspase-3的活化及细胞凋亡,其作用需要其激活结构域存在。AP-2α可能参与了COPD的发生发展。更多还原

【Abstract】 Background Chronic Obstructive Pulmonary Disease（COPD） is one of public health problems for its high morbidity and mortality,but its pathogenesis remains enigmatic and there is no good way to treat it. Cigarette smoke is one major etiologic factor of COPD.Besides chronic inflammation、the elastase/antielastase imbalance theory and oxidant/antioxidant imbalances,recently more and more clinical studies have shown apoptosis occurs in alveolar walls induced by cigarette smoking,resulting in progressive cell loss,emphysema and even respiratory failure.Thus,it is the key to interfere with apoptosis in the prevention and treatment of COPD.Transcription factor activator protein-2（AP-2） is a family of cell type-specific developmentally regulated transcription factors.To date,five members of the AP-2 family have been identified and AP-2αis the first one.AP-2αis shown to modulate the expressions of many genes such as VEGF、P⁵³、p^21WAF/CIP、Bcl-2 and Manganese superoxide dismutase（MnSOD）,and it has been implicated in vertebrate development、embryogenesis and carcinogenesis. Most of these studies have been focused on the regulation of apoptosis and proliferation.Depending on different cell type,AP-2αhas been shown to promote or inhibit apoptosis.As yet there are no related reports about the expression level of AP-2αin lung tissue from rat COPD models and human vascular endothelial cells ECV304 treated by CSE,and whether AP-2αexerts influence on apoptosis and the activated Caspase-3 induced by CSE,and whether its function is associated with its domain.Objective:To investigate the expression of AP-2αin COPD rat lung tissue and ECV304 cells stimulated by CSE,and furthermore to study the relationship between the expression of AP-2αand apoptosis in ECV304 cells stimulated by CSE and mechanisms.Methods:To replicate rat models of COPD by passive smoking.The apoptosis of alveolar septal cells was detected using Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling（TUNEL） methods and active Caspase-3 in the lung tissue was measured by western blot.The expression of AP-2αin rat lung tissue was detected by immunohistochemistry and Western blot.ECV304 cells were treated with different concentrations of CSE for indicated time,then tested for proliferation rate by MTT assay;Apoptosis rate by Hoechst 33258 nucleus staining;and Caspase-3 activation and AP-2αprotein levels by western blot.Mouse AP-2α、transactivation-domain mutated AP-2α（AP-2△） and respective control one were transfected into ECV304 cells by adenovirus-induced or plasmid transfection for 24 hours,then cells were treated with 5%CSE for another 24 hours.Cell proliferation rate,apoptosis rate and Caspase-3 activation levels were compared between the two groups. Results:Rat model of COPD were replicated successfully by passive smoking for eighty days.COPD group showed more inflammatory cells infiltration,larger air spaces and significant decrease of FEV_0.3/FVC compared with the normal group;The apoptosis rate of alveolar septal cells and the expression of active Caspase-3 in the lung tissue were increased indeed;the expression of AP-2αwas significantly higher than that in the controls（p＜0.05）.In ECV304 cell,5%,10%,15%and 20%CSE treatment for 24h significantly reduced cell proliferation rate（P＜0.005）,while 2.5% increased it.5%CSE treatment significantly increased Caspase-3 activation（p＜0.001） and ECV304 cell apoptosis shown by Hoechst 33258 nuclear staining（p＜0.01）.Also,5%CSE treatment increased endogenous AP-2αprotein expression in the ECV304 cells.AP-2αOver-expression in ECV304 cells inhibited 5%CSE-induced cell apoptosis and activated Caspase-3 expression（p＜0.05）,while the transactivation-domain mutated AP-2α（AP-2△） showed no protective effect on CSE-induced cell damage.Conclusions:It is concluded that CSE is one major etiologic factor of COPD;Apoptosis may be one of important mechanisms contributing to COPD;The expression of AP-2αwas significantly higher in lung tissue in COPD group and ECV304 cell stimulated by CSE than that in the controls.AP-2αcan protect ECV304 cell against CSE-induced cell damage and its activation domain is necessary for this function.更多还原