【作者】 刘佳；

【导师】 吴泰华；

【作者基本信息】 大连医科大学 ， 内科学， 2009， 硕士

【摘要】 背景:间质性肺疾病/肺纤维化是严重威胁人类健康和生命的一组疾病,其发病率呈逐渐上升趋势,死亡率高。其病理表现为肺间质炎性细胞的浸润,成纤维细胞的增生和分化,大量细胞外基质的聚集,最终导致肺结构的重塑。由于发病机制不十分清楚,目前尚无有效的治疗方法。虽然肺组织已形成的纤维化不可逆转,但肺间质纤维化是一个慢性的,进行性的病理反应过程,探索肺间质纤维化发病的分子机制及新的干预靶点以延缓或阻止肺纤维化进展的策略,仍具有重要的实际意义,一直是国际肺科学界的关注焦点。大量研究显示,在肺纤维化的发展过程中,转化生长因子-β(Transforming growth factorβ,TGF-β)起着关键作用。活化的TGF-β,在Ⅲ型受体的调节作用下,首先与肺成纤维细胞膜上的2个Ⅱ型受体结合,再募集2个Ⅰ型受体形成复合体,磷酸化Ⅰ型受体的GS功能域,使之活化,然后活化的Ⅰ型受体再磷酸化相应的下游信号分子Smads蛋白来发挥生物学效应。但具体哪几个TGF-βⅠ型受体亚型参与肺纤维化的过程尚不清楚。因此我们研究TGF-βⅠ型受体亚型在正常肺组织和肺间质纤维化发生和发展中的分布和变化规律,筛选出在肺间质纤维化中高表达的受体亚型,以求达到延缓或阻止肺间质纤维化疾病的作用。目的:明确TGF-βⅠ型受体亚型在正常肺组织和肺间质纤维化中的分布和变化规律;探讨特异性TGF-βⅠ型受体亚型在肺间质纤维化中的表达,综合的分析TGF-βⅠ型受体亚型在肺间质纤维化发生和发展中的分布和变化规律,为防治肺间质纤维化提供新的理论依据。方法:制作肺纤维化的动物模型,通过实时荧光定量PCR(Real Time-PCR)技术来检测TGF-βⅠ型受体亚型在正常肺组织和肺间质纤维化发生和发展中的分布和变化规律,结合肺组织的病理改变,筛选出有意义的TGF-βⅠ型受体亚型。结果:1. HE染色的病理切片观察HE染色可见各造模组均有程度不等的肺泡炎,肺泡腔及肺间质内有不等的炎性细胞浸润。7天组见炎症细胞浸润最明显,14天组见成纤维细胞增多。28天组见胶原纤维增生,部分肺泡腔萎缩消失。35天组见胶原纤维增生更明显。2. Real Time-PCR结果显示:与正常组相比,模型组纤维化大鼠肺组织中TGF-βⅠ型受体ALK-4、ALK-5和ALK-7mRNA的表达于第1天开始上升并随纤维化时间的延长逐渐增高,ALK-4mRNA于第14天达到最高峰,在第28天有所回落,而ALK-5和ALK-7mRNA于第28天达到最高峰,在第35天有所回落。TGF-βⅠ型受体ALK-1和ALK-2mRNA于第一天升高,而后随着纤维化时间的延长呈下降趋势。ALK-3和ALK-6的mRNA表达水平于第1天开始逐渐上升,分别于第7天和第14天达到高峰,而后逐渐下降。运用重复测量资料的方差分析,各TGF-βⅠ型受体亚型随时间点的变化比较均为P<0.05,有统计学意义。结论:1.ALK1-7均参与肺间质纤维化发生发展的过程。2.ALK-4、ALK-5、ALK-7在肺间质纤维化发生发展过程中呈高表达,相对浓度超过正常5倍以上。更多还原

【Abstract】 Background:Interstitial lung disease /pulmonary fibrosis is a serious risk to human health, the incidence rate is in a gradually ascending trend. The pathological process includes inflammation cells invading, collagen depositing, amounts of excellular matrix assembling in the mesenchyma, resulting in reformation of the lung structure. At present, there is no effective treatment in term of the unknown etiology and unclear pathogenesis. Although it is difficult to eliminate formation of pulmonary fibrosis, pulmonary fibrosis is a chronic, progressive pathological process. The strategies by exploring the molecular mechanism of the pathogenesis and new intervention target in delaying or blocking the progression of pulmonary fibrosis still have important practical significance and have been always a focus for the pneumo-science circle .TGF-βis a key cytokine in fibrosis. The definite mechanism is that activated TGF-βbinding to the typeⅡreceptor (RⅡ) leads to recruitment of the typeⅠreceptor (RⅠ) and formation of the receptor heterocomplex. In the complex, the constitutively active typeⅡreceptor phosphorylates the typeⅠreceptor at GS domain and actives it. The activated typeⅠreceptor is then able to bind and phosphorylate Smads protein. But the effect and mechanism of TGF-βⅠtype receptor and subtypes has not been understood. So the purpose of the study is to sieve the specific subtypes in the lung fibrosis. In this study, we explore the specific subtypes that exist in normal lung and pulmonary fibrosis by analyzing the distribution and changes of the subsets of TGF-βⅠR,to delay or block pulmonary fibrosis .Objective:To identify the distribution and expressive changes of the subsets of TGF-βⅠR in normal lung and pulmonary fibrosis. To explore the specific subtypes in pulmonary fibrosis, analysis the distribution and changes of the subsets of TGF-βⅠR in normal lung and pulmonary fibrosis synthetically and provide the new evidence for curing the lung fibrosis.Method:We established fibrosis models, identified the distribution and expressive changes of the subsets of TGF-βⅠR in normal lung and pulmonary fibrosis through Real Time-PCR, and selected the specific subtypes in the lung fibrosis combining with pathology.Result:1. Pathologic change by HE staining:HE staining showed that various kinds of inflammatory cells infiltrated in alveolus and pulmonary mesenchyme, the most obvious in the 7th group. Lung fibroblast began to increase in the 14th group. The fibrosis was clearer in the 28th group, the clearest in the 35th group, with alveolus atrophy and destruction.2. Real Time-PCR:Compared with normal group, the mRNA levels of ALK-4、ALK-5 and ALK-7 were significantly increased in the rat model of fibrosis established, but the ones of ALK-1 and ALK-2 were not marked.The mRNA levels of ALK-3 and ALK-6 were increased in the early stage and then inclined in the process of lung fibrosis.Using repeated measurements analysis of variance, comparing the expressive changes between groups at different time point, we concluded that the results were P<0.05 significantly.Conclusion:1. ALK1-7 participated in the process of lung fibrosis.2. The expression of ALK-4、ALK-5 and ALK-7 was more obvious than other subtypes in the process of lung fibrosis.Their relative concentrations were raised up to more than five times.更多还原