【作者】 牛瑞超；

【导师】 罗百灵；

【作者基本信息】 中南大学 ， 内科学， 2008， 博士

【摘要】 慢性阻塞性肺疾病（Chronic obstructive pulmonary disease,COPD）是一组严重危害人群健康的常见疾病,近年来,其发病率在全球均有明显上升趋势。COPD发病机制尚未完全阐明,目前多数学者认为弹性蛋白酶-抗蛋白酶失衡是COPD发生、发展的重要机制之一。研究表明,慢性支气管炎患者（无论急性发作与否）其气道腔内中性粒细胞百分数显著高于对照组。同时发现,慢支患者的气流阻塞程度与支气管灌洗液中中性粒细胞数量增多有关。以上研究表明,COPD患者气道腔内存在中性粒细胞的聚集状态,中性粒细胞弹力酶（NE）是中性粒细胞释放的丝氨酸蛋白酶,被认为是肺炎性损伤级联反应的主要终效应因子,其对肺组织结构蛋白有直接损伤作用,是肺气肿形成过程中的关键作用环节。中性粒细胞聚集导致弹性蛋白酶负荷的增加,从而促进了COPD的发生发展。与蛋白酶相对应,体内的抗蛋白酶主要有:1.α1-抗胰蛋白酶（α1-AT）,这是肺实质中主要的抗蛋白酶,它由肝脏合成,血浆转运。α1-AT主要对抗中性粒细胞弹性蛋白酶。2.α1-抗糜蛋白酶。3.组织金属蛋白酶抑制物（TIMPs）。该酶主要对抗MMPs。4.分泌性白细胞蛋白酶抑制物（secretory leukocyte proteinase inhibitor,SLPI）,这是气道中最重要的保护基质,它起源于气道上皮细胞,在气道中可起到局部防护作用。国内外研究最多、最透彻的是α1-AT,它是人血浆中最主要的抗蛋白酶,但其并不能完成在肺间质中的保护作用;相对而言,目前国内外对源于气道上皮细胞在气道中起到局部最重要防护作用的SLPI的研究却较少,SLPI能够抑制金属蛋白酶的产生,抑制核因子-κB（NF-κB）的活性和减轻炎症反应。SLPI还可以抑制气道上皮细胞中性粒细胞弹性蛋白酶（NE）的水平和白细胞介素8（IL-8）的水平,从而可以中断炎症循环,提示SLPI可能是气道中最重要的保护基质之一。转化生长因子β₁（TGF-β₁）也被认为是COPD发病机制中的关键致病因子,其作用机制可能与肺纤维化的形成及气道重构密切关联。已有研究证实,TGF-β受体激活后,由TGF-βⅠ型受体到核内的信号转导主要由Smads蛋白家族的胞浆蛋白磷酸化完成的。被激活的已经磷酸化的Smad2和Smad3只有与Smad4结合形成复合物,才能使TGF-β₁信号传导进入细胞核,从而起到生物学效应。然而,在COPD发病过程中,SLPI在支气管肺组织中的如何表达及其是否与TGF-β₁存在对应关系尚不清楚,若二者存在对应关系,Smads信号通路是否在此过程中起作用作用。本研究对此进行了探讨。第一部分:分泌性白细胞蛋白酶抑制物在慢性阻塞性肺疾病大鼠支气管肺组织中的表达及转化生长因子β₁对其影响的研究我们采用熏香烟加气管内注射内脂多糖复制大鼠COPD模型,并用TGF-β₁单抗干预,测定大鼠的肺功能及病理变化结果,应用酶联免疫法检测支气管肺泡灌洗液（BALF）中SLPI的水平,用免疫组织化学法观察TGF-β₁、Smad 4、SLPI在支气管肺组织中的表达情况,用逆转录聚合酶链法检测TGF-β₁、Smad4和SLPI mRNA在支气管肺组织中的表达水平。结果显示:光镜下可见模型组支气管上皮细胞变性坏死脱落,大量炎症细胞浸润,肺大疱形成,气管及血管壁增厚,管腔狭窄闭塞。干预组可见少量支气管上皮细胞变性坏死脱落,气管及血管壁轻度增厚,少量炎性细胞浸润,管壁未见明显增厚,未见明显肺大疱的形成及管腔狭窄。而正常对照组未见异常。模型组大鼠比对照组大鼠的肺功能明显降低,干预组大鼠比模型组大鼠的肺功能明显增高。TGF-β₁在血管和气道平滑肌细胞、支气管、细支气管上皮细胞、肺泡上皮细胞、肺间质细胞以及巨噬细胞均有免疫染色,细胞膜着色。对照组TGF-β₁呈弱阳性表达,阳性产物为棕黄色颗粒,模型组TGF-β₁呈强阳性表达,阳性产物为棕褐色颗粒,二者的阳性系数和灰度值差异有统计学意义;干预组TGF-β₁表达比模型组明显减弱,二者的阳性系数及灰度值差异有统计学意义。Smad4在血管和气道平滑肌细胞、支气管、细支气管上皮细胞、肺泡上皮细胞、肺间质细胞以及巨噬细胞均有免疫染色,细胞质和细胞核均有着色。对照组Smad4呈弱阳性表达,阳性产物为棕黄色颗粒,模型组Smad4呈强阳性表达,阳性产物为棕褐色颗粒,二者的阳性系数和灰度值差异有统计学意义;干预组比模型组Smad4表达明显减弱,二者的阳性系数和灰度值差异有统计学意义。SLPI只在气管、支气管上皮细胞有免疫染色,且仅在细胞质和管腔侧细胞膜有着色。对照组SLPI呈强阳性表达,阳性产物为棕褐色颗粒,模型组SLPI呈弱阳性表达,阳性产物为棕黄色颗粒,二者的阳性系数和灰度值差异有统计学意义;干预组比模型组SLPI表达明显增强,二者的阳性系数和灰度值差异有统计学意义。模型组支气管肺泡灌洗液中SLPI浓度明显低于对照组,差异有统计学意义;干预组支气管肺泡灌洗液中SLPI浓度明显高于模型组,差异有统计学意义。与对照组比较,模型组大鼠支气管肺组织中TGF-β₁、Smad4 mRNA表达量增高,其吸光度值明显增高,差异有统计学意义;而SLPI mRNA的表达量却很低,其吸光度值明显降低,差异有统计学意义。干预组大鼠支气管肺组织中TGF-β₁、Smad4 mRNA表达量比模型组明显下降,其吸光度值明显降低,差异有统计学意义;而SLPI mRNA的表达量却明显升高,其吸光度值明显增高,差异有统计学意义。第二部分:Smads信号通路在TGF-β₁介导气道上皮细胞SLPI下调中作用的研究我们采用Smad4 siRNA预处理及TGF-β₁进行刺激支气管上皮细胞,免疫细胞化学及westen blot方法观Smad4、SLPI在Smad4 siRNA预处理及TGF-β₁进行刺激后支气管上皮细胞内的表达情况;RT-PCR法检测在Smad4 siRNA预处理及TGF-β₁进行刺激后支气管上皮细胞内Smad4 mRNA及SLPI mRNA的表达水平。结果显示:SLPI在胞浆胞膜均有免疫染色,在对照组中SLPI呈强阳性表达,染色为棕褐色颗粒,经TGF-β₁刺激后,SLPI在刺激组呈弱阳性表达的棕黄色颗粒;Western blot检测也显示,在对照组中SLPI表达量较高,经TGF-β₁刺激后,SLPI在刺激组表达量明显减少,二者差异差异有统计学意义;在对照组中SLPI mRNA表达量较高,经TGF-β₁刺激后,SLPI mRNA在刺激组表达量明显降低,二者差异有统计学意义。Smad4在胞浆胞核均有免疫染色,在对照组中Smad4呈弱阳性表达,染色为棕黄色颗粒,经TGF-β₁刺激后,Smad4在刺激组染色呈强阳性表达的棕褐色颗粒;Western blot检测也显示,在对照组中Smad4表达量较低,经TGF-β₁刺激后,Smad4在刺激组表达量明显增高,二者差异有统计学意义;在对照组中Smad4 mRNA表达量较低,经TGF-β₁刺激后,Smad4 mRNA在刺激组表达量明显增高,二者差异有统计学意义。经Smad4 siRNA预处理后再给予TGF-β₁刺激,Smad4在干扰组的表达比单纯的TGF-β₁刺激组的表达明显减弱,染色呈弱阳性表达的棕黄色颗粒;而给予阴性对照siRNA预处理后再给予TGF-β₁刺激,则Smad4在HK组的表达比单纯的TGF-β₁刺激组的表达减弱不明显,染色仍呈强阳性表达的棕褐色颗粒;Western blot检测也显示,经Smad4 siRNA预处理后再给予TGF-β₁刺激,Smad4在干扰组的表达量比单纯的TGF-β₁刺激组明显减少,二者差异有统计学意义。而给予阴性对照siRNA预处理后再给予TGF-β₁刺激,则Smad4在HK组的表达量比单纯的TGF-β₁刺激组减少不明显,二者差异无统计学意义。经Smad4 siRNA预处理后再给予TGF-β₁刺激,Smad4 mRNA在干扰组的表达量比单纯的TGF-β₁刺激组明显减少,二者差异有统计学意义;而给予阴性对照siRNA预处理后再给予TGF-β₁刺激,则Smad4 mRNA在HK组的表达量比单纯的TGF-β₁刺激组的表达量减少不明显,二者差异无统计学意义。经Smad4 siRNA预处理成功转染后再给予TGF-β₁刺激,SLPI在干扰组的表达比单纯的TGF-β₁刺激组的表达明显增强,染色呈强阳性表达的棕褐色颗粒;而给予阴性对照siRNA预处理后再给予TGF-β₁刺激,则SLPI在HK组的表达比单纯的TGF-β₁刺激组的表达增强不明显,染色仍呈弱阳性表达的棕黄色颗粒;Western blot检测也显示,经Smad4 siRNA预处理后再给予TGF-β₁刺激,SLPI在干扰组的表达量比单纯的TGF-β₁刺激组明显增高,二者差异有统计学意义;而给予阴性对照siRNA预处理后再给予TGF-β₁刺激,则SLPI在HK组的表达量比单纯的TGF-β₁刺激组增高不明显,二者差异无统计学意义。经Smad4 siRNA预处理成功转染后再给予TGF-β₁刺激,SLPImRNA在干扰组的表达量比单纯的TGF-β₁刺激组明显增高,二者差异有统计学意义;而给予阴性对照siRNA预处理后再给予TGF-β₁刺激,则SLPI mRNA在HK组的表达量比单纯的TGF-β₁刺激组增高不明显,二者差异无统计学意义。上述二个部分研究提示,COPD大鼠支气管肺组织中SLPI明显减少,可能主要是由于TGF-β₁表达增加所致,而且,TGF-β₁使支气管上皮细胞SLPI表达下调的作用是通过Smads信号通路介导的。更多还原

【Abstract】 Chronic obstructive pulmonary disease（COPD）is characterized by airflow obstruction,which comprises emphysema and chronic bronchitis/bronchiolitis.While the molecular mechanisms by which small airway obstruction occurs remain unknown.Recently,some studies indicated that an imbalance between neutrophil protease and surrounding antiprotease levels has been shown to be important in the pathogenesis of COPD.Secretory leukocyte proteinase inhibitor（SLPI）,known as one of the most important antileukoproteases in airway,is a 12-kDa non-glycosylated,cationic protein that is produced by serous cells of the submucosal bronchial glands,by non-ciliated cells of the bronchial epithelium,and by neutrophils.Its major physiological function is considered to be the inhibition of the destructive capacity of neutrophil elastase（NE）,and some data indicated that COPD is related to the reduction of SLPI.Whereas little is known about the regulation of SLPI expression in the lung.Other studies suggest some fibrogenic growth factors may be involved in the remodeling processes of the small airways,iOne of the most potent and extensively studied growth factors is transforming growth factor（TGF）-betal,which induces fibroblast proliferation, increased production of collagen and other extracellular matrix proteins, and decreased collagen degradation.TGF-β₁ is also chemotactic for neutrophil,macrophages and mast cells,and its major intracellular signaling effector is the Smad proteins.It is well known that Smad pathways are central mediators of signals from the receptors for TGF-beta superfamily members to the nucleus.Phosphorylation of receptor-activated Smads（R-Smads）leads to formation of complexes with the common mediator Smad（Co-Smad）（Smad4）,which are imported to the nucleus.Nuclear Smad oligomers bind to DNA and associate with transcription factors to regulate expression of target genes. TGF-β₁ is widely localized in the lung.Several studies recently demonstrated that there was a significant expression of TGF-β₁ in airway epithelial cells in subjects with COPD as compared with the control.Low levels of SLPI and high levels of TGF-β₁ were observed in the bronchi and lung tissues of COPD,and recent study indicated that TGF-β₁ is a potent inhibitor of SLPI in a bronchial epithelial cell. However,whether the decreased expression of SLPI in the bronchi and lung tissues of COPD is related to the increased expression of TGF-β₁ is unknown.If so,whether the role of SLPI is mediated through Smads signal pathway needs further investigation.To address the role of TGF-β₁/Smads on the regulation of SLPI in the bronchi and lung tissues of COPD,COPD model was established and the rats were treated with TGF-β₁ monoclonal antibody;the normal human bronchial epithelial cell （NHBE）line was cultured and it was stimulated with TGF-β₁ and siRNA （Smad4）.Then,the relationship among the expression of SLPI TGF-β₁ and Smad4 in the bronchi and lung tissues were observed,and the role of TGF-β₁/Smads on the decreased expression of SLPI in NHBE cells was investigated.1.Expression of secretory leukocyte proteinase inhibitor in the bronchi and lung tissues of chronic obstructive pulmonary disease rat models and the regu-lative mechanism by TGF-β₁Rat COPD model was established by intratracheal instillation of lipopoly-saccharide（LPS）twice and exposure to cigarette smoke daily; drug intervention group received TGF-β₁ monoclonal antibody 0.5 mg twice via the tail venous injection.The spirometry was conducted and the pathological changes were observed,the concentrations of SLPI in bronchoalveolar lavage fluid（BALF）was measured by enzyme-linked inmunosorbent assay（ELISA）;The expressions of TGF-β₁、Smad4 and SLPI in the bronchi and lung tissues were examined by using immunohistochemistry,and the expressions of TGF-β₁ mRNA,Smad4 mRNA and SLPI mRNA in the bronchi and lung tissues were detected by reverse transcription-polymerase chain reaction（RT-PCR）respectively.Results showed:The PEF,FEV_0.3and FEV_0.3/FVC in COPD model group were significantly lower than those in the control group（all P＜0.01）.After treated with TGF-β₁ monoclonal antibody,the PEF,FEV_0.3 and FEV_0.3/FVC in the TGF-β₁ monoclonal antibody intervention group were all significantly improved as compared with the COPD model group （all P＜0.01）.Compared with the control group,a portion of the airway epithelium and some cilia had been shed,inflammatory cells infiltrated some airway walls,smooth muscles in the airway walls and small artery walls had proliferated irregularly,air spaces were enlarged in an irregular manner,some alveoli were confluent and bullae were seen in the COPD model group.Compared with the COPD model group,significant improvement was seen in the shedding of airway epithelium and cilia,the proliferation of smooth muscle in airway walls and small artery walls was great alleviated,air spaces were not obviously enlarged in the TGF-β₁ monoclonal antibody intervention group.The concentration of secretion of SLPI in BALF of the COPD model group was significantly lower than that in the control group（P＜0.01）.After treated with TGF-β₁ monoclonal antibody,the concentration of secretion of SLPI in BALF of the TGF-β₁ monoclonal antibody intervention group was significantly improved as compared with the COPD model group（all P＜0.01）.Both at the mRNA level and the protein level,the expressions of both TGF-β₁ and Smad4 in the COPD model group were higher than those in the control group（all P＜0.01）,while the expression of SLPI was lower in the COPD model group than that in the control group（P＜0.01）.After treated with TGF-β₁ monoclonal antibody,the expressions of both TGF-β₁ and Smad4 were all significantly decreased in TGF-β₁ monoclonal antibody intervention group both at the mRNA level and the protein level as compared with the COPD model,grouP（all P＜0.01）,however,the expression of SLPI was significantly increased in TGF-β₁ monoclonal antibody intervention group both at the mRNA level and the protein level as compared with the COPD model group（all P＜0.01）.According to TGF-β₁ and Smad4,the main immune positive cell types were vascular smooth muscle cell, airway epithelial cell,alveolar epithelial cell,pulmonary interstitial cell and macrophage cells;but to SLPI,the main immune positive cell type was only airway epithelial cell.TGF-β₁ was mainly stained in membrane, Smad4 and SLPI were both stained in cytoplasm,but Smad4 was stained in nuclus,too.2.The expression of secretory leukocyte proteinase inhibitor in human bronchial epithelial cell is downregulated by TGF-β₁/Smads pathway.The normal human bronchial epithelial cell（NHBE）was cultured, preincubated with or without siRNA（Smad4）,and then stimulated with or without TGF-β₁.The expressions of Smad4 and SLPI were detected by immunocytochemistry,western blot and RT-PCR respectively.Results showed:The expression of SLPI in NHBE was inhibited by TGF-β₁ both at the mRNA level and the protein level（all P＜0.01）.The expression of Smad4 in NHBE was successfully inhibited by siRNA （Smad4）both at the mRNA level and the protein level（all P＜0.01）,but it was not affected by negative control siRNA（all P＞0.05）.At the same time,the results indicated that,after preincubated with siRNA（Smad4） then stimulated with TGF-β₁,the effects of TGF-β₁-inhibited expression of SLPI in NHBE was disengaged by siRNA（Smad4）both at the mRNA level and the protein level（all P＜0.01）,but it was not affected by negative control siRNA.（all P＞0.05）The above two parts prompted:the expression of SLPI in the airway of the COPD rat model was significantly decreased,which may be mainly caused by the increased expression of TGF-β₁,and the activation of Smads signal pathway play a crucial role in this process.更多还原