DDR2在肺纤维化病理进程中作用的研究

发布时间：2018-03-03 21:22

本文选题：DDR2　切入点：肺纤维化　出处：《第四军医大学》2012年硕士论文　论文类型：学位论文

【摘要】：肺纤维化是由多种因素引起的慢性肺间质性疾病。由于工业的高速发展、吸烟人群以及老龄化人口的增加，肺纤维化已成为我国的常见病和多发病。尽管科学家对此疾病已经进行了大量的研究，但到目前为止，肺纤维化的病因和发病机制仍然不甚清楚。早期诊断困难、预后差以及缺乏有效的治疗手段使得肺纤维化成为难以攻克的疾病，亟待更深入的研究。 DDR2分子属于酪氨酸蛋白激酶受体（Receptor Tyrosine Kinase，RTK）家族成员，介导细胞与细胞外基质之间的信号转导，广泛参与细胞增殖分化、个体发育及生殖等生理病理过程。当配体与DDR2结合后，其胞内区的酪氨酸残基发生磷酸化。磷酸化的位点会进一步招募下游的信号分子，最终导致整个信号通路的活化。其活化的持续时间较经典RTK明显提高，提示DDR2可能参与体内较缓慢的病理过程。已有研究表明，DDR2仅在肺、胃等脏器中高表达。而肺纤维化过程中产生的大量胶原，特别是I型和III型胶原，，为活化DDR2提供了空间上的可能；在人原发性胆汁性肝硬变和大鼠酒精性肝纤维化模型中，DDR2的表达水平随着病程的发展而逐渐升高；DDR2还能通过调控胶原酶MMP-1和MMP13的表达参与类风湿性关节炎的发生发展过程，而MMPs正好是肺纤维化的重要参与分子。因此，我们推测DDR2在肺纤维化过程中必然发挥重要作用。我们主要进行了以下几方面的研究： 1.在人正常肺标本和肺纤维化标本中通过免疫荧光方法检测DDR2的表达差异。 2.建立小鼠Bleomycin肺纤维化模型，实时定量PCR和Western Blot方法检测肺纤维化过程中的DDR2表达变化。 3.在DDR2基因缺失小鼠上建立肺纤维化动物模型，HE染色、Masson染色、ELISA、免疫荧光、TUNEL、明胶酶谱等方法明确DDR2在肺纤维过程中的作用。 4. siDDR2特异性下调野生鼠肺组织DDR2的表达，采用HE、Masson等方法观察DDR2被干涉之后肺纤维化水平变化。 5. TGF-β1刺激小鼠原代肺成纤维细胞，实时定量PCR和Western Blot方法检DDR2信号阻断后纤维化相关分子以及下游信号分子的表达变化。 6.在肺纤维化模型上，观察DDR2特异性小分子抑制剂对肺纤维化的影响。结果：人肺纤维化标本较正常肺标本DDR2表达水平明显升高；小鼠肺纤维化过程中DDR2表达呈现先下降后上升的趋势；DDR2基因缺失后炎症侵润现象明显减轻，MMPs表达和分泌下降；DDR2野生鼠中肺纤维化水平明显升高，肺组织羟脯氨酸含量和肺泡灌洗液中TGF-β1的量明显高于DDR2基因缺失鼠，纤维化晚期凋亡细胞也较野生鼠明显减少；细胞学实验表明肺纤维化相关分子的表达在DDR2缺失后随着TGF-β1诱导时间的延长显著下降，AKT、p38等下游信号分子磷酸化水平和肌成纤维细胞数目也显著下降。
[Abstract]:Pulmonary fibrosis is a chronic pulmonary interstitial disease caused by many factors. Pulmonary fibrosis has become a common disease and frequent disease in China. Although scientists have done a lot of research on this disease, the etiology and pathogenesis of pulmonary fibrosis are still unclear. Poor prognosis and lack of effective treatment make pulmonary fibrosis a difficult disease. DDR2 molecule is a member of the tyrosine protein kinase receptor receptor receptor (DDR2). It mediates the signal transduction between cells and extracellular matrix, and participates in many physiological and pathological processes, such as cell proliferation, differentiation, ontogenesis and reproduction. When ligand binds to DDR2, it plays an important role in the process of cell proliferation, differentiation, ontogenesis and reproduction. The phosphorylation of tyrosine residues in the intracellular region may further recruit downstream signaling molecules and eventually lead to activation of the entire signaling pathway. The duration of activation is significantly longer than that of classical RTK. The results suggest that DDR2 may be involved in the slower pathological process in vivo. It has been shown that DDR2 is only highly expressed in the lung and stomach. However, the large amount of collagen produced in the process of pulmonary fibrosis, especially type I and III collagen, provides a spatial possibility for activating DDR2. The expression of dddR2 in human primary biliary cirrhosis and alcoholic liver fibrosis model increased gradually with the development of the course of the disease. The expression of DDR2 also participated in the pathogenesis of rheumatoid arthritis by regulating the expression of collagenase MMP-1 and MMP13. Therefore, we speculate that DDR2 must play an important role in the process of pulmonary fibrosis. We have mainly carried out the following studies:. 1. The expression of DDR2 was detected by immunofluorescence in normal lung samples and pulmonary fibrosis specimens. 2. The mouse Bleomycin pulmonary fibrosis model was established, and the expression of DDR2 in pulmonary fibrosis was detected by real-time quantitative PCR and Western Blot. 3. The animal model of pulmonary fibrosis was established in mice with DDR2 gene deletion. The role of DDR2 in the process of pulmonary fiber was clarified by means of HE staining and Masson staining, immunofluorescence Tunel and gelatinase spectrum. 4. SiDDR2 specifically down-regulated the expression of DDR2 in the lung tissues of wild rats, and observed the changes of pulmonary fibrosis after DDR2 was interfered with. 5. TGF- 尾 1 stimulated primary mouse lung fibroblasts, and real-time quantitative PCR and Western Blot were used to detect the expression of fibrosis related molecules and downstream signal molecules after DDR2 signal was blocked. 6. To observe the effect of DDR2 specific small molecule inhibitor on pulmonary fibrosis model. Results: the expression of DDR2 in human lung fibrosis was significantly higher than that in normal lung. In the course of pulmonary fibrosis in mice, the expression of DDR2 decreased first and then increased. The expression and secretion of DDR2 in the wild mice decreased significantly after the absence of DDR2 gene. The contents of hydroxyproline in lung tissue and TGF- 尾 1 in alveolar lavage fluid were significantly higher than those in mice without DDR2 gene, and the apoptotic cells in the late stage of fibrosis were significantly lower than those in wild mice. Cytological experiments showed that the expression of pulmonary fibrosis related molecules decreased significantly with the prolongation of TGF- 尾 1 induction time after DDR2 deletion, and the phosphorylation level of downstream signal molecules such as AKTTN p38 and the number of myofibroblasts also decreased significantly.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R563.9

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