【作者】 倪莲芳；

【导师】 刘新民；

【作者基本信息】 北京大学 ， 老年内科学， 2008， 博士

【摘要】 肺纤维化是一组由多种病因所引起的间质性肺疾病,是许多慢性肺病的共同结局。其病理特点包括肺泡上皮损伤、肺成纤维细胞积聚以及细胞外基质的过度沉积,最终导致不可逆性损伤。肺纤维化病因复杂、发病机制尚不明确。近年研究表明,免疫机制参与了肺纤维化的发病。主要组织相容性抗原(MHC)II类分子是抗原呈递细胞将外源性抗原提呈给CD4+Th细胞的必要载体,同时影响着T细胞的选择、免疫耐受的诱导及抗体产生等,是机体免疫反应的关键环节。II类反式激活蛋白(CIITA)是MHC II类分子表达的限速因子,人白细胞抗原(HLA)-DR是经典的MHC II类抗原。MHC II类抗原在靶器官的异常表达是组织特异性炎症免疫性疾病的重要标记。研究表明,MHC II类分子参与了肝、肾纤维化的发病过程。MHC II类分子参与肝脏纤维化的机制可能是MHC II类分子相关的抗原呈递导致B细胞的分化,由此产生高免疫球蛋白血症;而在肾脏则认为与MHC II类分子的抗原呈递作用刺激初始T细胞的特异性免疫应答,Th1/Th2极化失衡有关。MHC II类分子与肺纤维化之间的关系目前尚不清楚。目的:在博来霉素诱导的大鼠肺纤维化模型上,观察肺组织MHC II类分子及其上游调控因子CIITA表达水平的变化,临床实验观察HLA-DR在特发性肺纤维化的表达,探讨MHC II类分子在肺纤维化发病中可能的病理生理意义。方法:(1)雄性Wistar大鼠20只,随机分为对照组、肺纤维化组(经气管内注入BLMA5 5mg/kg),分别于造模后第7天、28天取肺组织行HE染色、Masson染色,并依据szapiel方法对肺部病变进行半定量评分;生化方法检测肺组织羟脯氨酸含量;(2)免疫组化染色观察大鼠肺组织MHC II类分子表达,实时定量PCR技术测定肺组织总CIITA及I型、III型、IV型CIITAmRNA和TGF-β1mRNA表达;(3)ELISA法检测大鼠肺泡灌洗液中IFN-γ及IL-4水平变化;(4)收集肺活检的特发性肺纤维化(IPF)患者肺组织石蜡标本10例,免疫组化染色观察肺组织HLA-DR的表达情况。结果:(1)气管内给予博莱霉素后第7天及第28天,大鼠肺组织MHC II阳性细胞明显增多(P<0.05, P<0.01),第28天较第7天增多(P<0.05);(2)第7天时,纤维化组大鼠肺组织总CIITA较对照组升高170.4%(P<0.05),I型CIITA较对照组升高258.8%(P<0.05),IV型CIITA较对照组降低87.2%(P<0.01);第28天时,纤维化组大鼠肺组织总CIITA较对照组升高98.6%(P<0.05),I型CIITA较对照组升高137.1%(P<0.05),IV型CIITA仍较对照组低,但差异无统计学意义;纤维化组肺组织IV型CIITA在第28天时较第7天时升高667.3%(P<0.01);(3)纤维化组肺组织第7天TGF-β1mRNA水平升高,达对照组的8倍多(P<0.01),第28天TGF-β1mRNA较第7天略有下降,但仍显著高于对照组(P<0.01),约为对照组的4倍;(4)纤维化组大鼠第7天及第28天BALF中IFN-γ含量均较对照组下降(均P<0.01),而同时期BALF中IL-4含量均较对照组升高(均P<0.01);(5)IPF肺组织细支气管肺泡上皮HLA-DR表达上调,与对照组比较差异具有统计学意义(Z=-3.002, P=0.001)。结论:肺组织MHCII/CIITA体系参与了博莱霉素诱导的肺纤维化的病理生理过程,这可能是通过调节肺组织局部的Th1/Th2平衡实现的;纤维化肺组织上皮细胞异常表达MHC II类抗原,推测这可能使这些上皮组织更易遭受MHC II类分子介导的免疫损伤,导致上皮损伤及异常修复的持续存在,最终导致肺纤维化形成并持续进展。更多还原

【Abstract】 Pulmonary fibrosis (PF) is the final common pathway of a diverse group of lung disorders known as a devastating disease. It’s characterized by epithelial injury, the formation of distinctive fibroblast/myofibroblast foci, and excessive extracellular matrix accumulation. The mechanisms by which lung fibrosis develops are not fully ascertained. Recent studies proved that abnormality of immune function might play an important role in the development of PF. Major histocompatibility complex (MHC) class II molecules play a critical role in the induction and regulation of adaptive immune responses through presentation of processed antigen to CD4+T lymphocytes, and they contribute to the maintenance of self-tolerance, as well as to the breakdown of tolerance in autoimmune diseases. The class II transactivator CIITA is the key regulator for expression of MHC class II molecules. The highly regulated expression pattern of CIITA ultimately dictates the cell type specificity, induction and level of MHC class II expression. Human leukocyte antigen (HLA)-DR is the classical MHC class II antigen. MHC class II molecules, when aberrantly expressed on epithelial cells, result in the activation of autoreactive lymphocytes and play a primary role in the initiation of autoimmune disease. It has been proved that MHC class II molecules were involved in the development of hepatic and renal fibrosis in rats. In the liver, it presumes that antigen presentation by MHC class II molecules induces differentiation of B cells and acts as an accelerator of immunoglobulin production, and high immunoglobulinemia and deposition of immunoglobulin in the liver. And in the kidney, it is said that MHC class II expressing cells presents antigens to unprimed T cells, which determines Th1/Th2 lymphocyte polarization in turn. But to date, the role of MHC class II molecules in the pathogenesis of pulmonary fibrosis has not been reported.Objective: (1) To explore the expression levels of MHC class II molecules and its regulator gene CIITA on bleomycin-induced pulmonary fibrosis in rats. (2) To study the expression of HLA-DR molecules on lungs in patients with idiopathic pulmonary fibrosis.Methods: (1) Twenty male Wistar rats were randomly divided into control group, fibrosis group. The rats were either treated with a single intratracheal bleomycin injection (fibrosis group) or normal saline injection (control group). Four to six rats in each group were sacrificed 7 and 28 days after intratracheal instillation. Histological changes in the lungs were evaluated by hematoxylin-eosin and masson stain, and scored. Contents of hydroxyproline in lung tissues were detected. (2) The expression of MHC class II molecules in lung tissues was evaluated, and the percentage of MHC class II positive cells was measured. The amounts of total CIITA and type I, III and IV CIITA and TGF-β1 mRNA of lung tissues were measured by real-time PCR using Taqman probe. (3) Levels of IFN-γand IL-4 in bronchoalveolar lavage fluid (BALF) of rats were measured by enzyme-linked immunosorbent assay (ELISA). (4) HLA-DR antigens in lung specimens from 10 cases of idiopathic pulmonary fibrosis (IPF) were detected using immunohistochemical technology.Results: (1) The percentage of MHC class II positive cells in lung tissues increased significantly in fibrosis group compared with that of control group on the 7th day and 28th day(P<0.05, P<0.01, respectively); In fibrosis group, the percentage on day 28 was higher than that on day 7 (P<0.05); (2) Compared with control group on the 7th day, total CIITA mRNA increased 170.4%(P<0.05), type I CIITA mRNA increased 258.8%(P<0.05), while type IV CIITA mRNA decreased 87.2%(P<0.01); On the 28th day, total CIITA mRNA increased 98.6%(P<0.05), type I CIITA mRNA increased 137.1%(P<0.05), type IV CIITA mRNA still decreased, but there was no significant difference (P>0.05); In fibrosis group, type IV CIITA mRNA was 667.3%(P<0.01)higher on day 28 than that on day 7; (3) The amount of TGF-β1 mRNA increased significantly in fibrosis group compared with that of control group on day 7, 28(P<0.01, respectively); (4) The concentration of IFN-γin BALF of fibrosis group was significantly lower than that of control group on day 7, 28(P<0.01, respectively), yet the IL-4 of fibrosis group was significantly higher than that of control group(P<0.01, respectively); (5) The HLA-DR antigens were abnoamally expressed on hyperplastic bronchio-alveolar epithelial cells in IPF, but not in normal control lung tissues. The accumulated positive scores of HLA-DR was 27 in the IPF group and 2 in control group (Z=-3.002, P=0.001).Conclusion: (1) MHCII/CIITA system of lung tissues was involved in the bleomycin-induced rat pulmonary fibrosis. It might participate in the pathogenesis of lung fibrosis by regulating the Th1/Th2 balance in the lung. (2) Inappropriate MHC class II molecules expression was present in bronchio-alveolar epithelium in pulmonary fibrosis. Epithelial cells expressing class II MHC molecules might be better recognized by autoreactive CD4+T cells and serve as better targets for MHC class II mediated immunic injury, which might lead to repeated lung injury and abnormal repair and remodeling. This might lead to the formation of lung fibrosis and further progression.更多还原