【作者】 孙健；

【导师】 曾昭玉； 毛国根；

【作者基本信息】 浙江大学 ， 劳动卫生与环境卫生学， 2004， 硕士

【摘要】 【目的】 矽肺是我国危害最严重的职业病之一，目前其发病机制仍不明确。矽肺最基本的病理变化是肺间质纤维化和矽结节的形成。与矽肺的发病相似，特发性肺纤维化(IPF)也是以肺成纤维细胞大量增殖为主要特征的肺纤维化疾病。两者的发病有着共同的特征，即持续性的肺泡炎症反应，以及各种炎前介质和致纤维化前介质的产生。提示两者在肺纤维化的发病过程中可能具有同源性。然而，相对于IPF及其他肺纤维化疾病而言，矽肺除了肺纤维化改变以外，还有其特征性的矽结节的形成。 支气管肺泡灌洗液(BALF)中的蛋白成分来源于气道及肺泡上皮表层，它能反映肺实质的病变过程。由于上述IPF、矽肺纤维化、矽结节等发病机制的复杂性，近年来，BALF中可溶性蛋白的蛋白质组学研究受到了关注，有关肺纤维化相关机制的研究已上升到细胞分子水平。 为了进一步探索与肺纤维化发生有关的细胞因子，本实验应用蛋白质组的双向电泳技术，浙江大学硕士学位论文—中文摘要孙健2004.5.对正常、IPF及矽肺BALF中的可溶性蛋白成分进行分离。并通过比较分析找出差异蛋白的信息，为下一步筛选、寻找及鉴定差异表达蛋白奠定基础，从而为阐明肺纤维化、矽结节形成机制提供新的思路。 【方法】 选用本校实验动物中心提供的清洁级健康成年雄性SD大鼠，随机分成3组。分别以0.9%的生理盐水lml、sm叭g的博来霉素和50m留ml的5102混悬液lml气管内灌注，制备正常、特发性肺纤维化及矽肺动物模型。通过支气管肺泡灌洗术获取三组大鼠的BALF样品，Folin一酚法测定样品中的蛋白含量。采用三氯乙酸(TCA)沉淀法提取大鼠BALF中蛋白样品，基本过程:取上述BALF样品加入TCA，使样品溶液的TCA终浓度为10%;混匀后冰浴20分钟，3500rpm离心1 smin;弃上清液，沉淀物以预冷的丙酮溶解，再次35oorpm离心巧min;倒弃丙酮，蛋白沉淀物风干几分钟后，以水化上样缓冲液充分溶解，超声混匀，40c下300009离心30min，保留上清液即为大鼠BALF蛋白样品。利用蛋白质组学的双向电泳技术，按180林g蛋白加样量(300林l的水化液)，以17em长的PH为3一10的线性IPG胶条进行第一相等电聚焦，再以12%505一队GE作第二相分离，分别对三组BALF中的蛋白质成分进行分离。运用凝胶分析软件PDQuest对所获取三组大鼠BALF的双向电泳图谱进行比较分析。数据的统计分析采用SPSS 11.0进行处理。【结果】 1.大鼠肺组织常规病理切片的形态学观察 常规病理切片显示正常对照组肺组织结构完整，肺泡壁均匀、清晰;IPF组可见肺组 织结构基本完整，肺泡壁增宽，胶原成分增多;矽肺组可见肺组织结构出现不同程度的破 坏，并出现典型的矽结节的改变，矽结节以细胞成分为主，其内胶原纤维增加。肺间质纤 维组织增生。2.各组BALF样品中蛋白含量的比较 三组BALF样品中蛋白含量测定结果显示:正常组为o.3196mglml、!pF组为0.3075mglml、矽肺组为1 .2775mglml。经单因素方差分析差别有显著意义(F二15.293:P<0.001)。与正常对照组比较，IPF组日ALF中蛋白含量无明显的统计学差异，而矽肺组BALF中蛋白质含量增高(P二0.002)。3.大鼠BALF的蛋白质2一DE图谱浙江大学硕士学位论文—中文摘要孙健.2004.5. 采用双向电泳技术分离蛋白质，以17cm长的pH为3一10的线性IPG胶条进行第一 相等电聚焦，再以12%垂直SDS一队GE作为第二相分离，获得了较清晰的正常、特发性 肺纤维化及矽肺大鼠BALF中蛋白质的双向电泳图谱。使用数字化ZD凝胶图像分析软件 PDQuest对所获取的蛋白质斑点进行分析，共获取蛋白斑点数为(450士63.2)个，其中正常 对照组的斑点数为(488士62.0)个，IPF组蛋白斑点为(469士42.2)个，矽肺组的蛋白质斑点 为(3 93士51.9)个。在所有的胶中共有123.3个蛋白斑点获得了完全匹配，而有349个蛋白 质斑点可以获得相对匹配。对同组的图谱进行匹配分析，在正常对照组共有387个点获得 了匹配，IPF组有355个点获得匹配，而矽肺组有306个点获得匹配。三组样品蛋白匹配 率分别为77.6%，75.3%及77%，获得较好的匹配。4.正常、IPF及矽肺大鼠BALF中蛋白质表达的差异 对三组样品中蛋白质差异斑点进行分析，发现其部分蛋白质斑点出现了质的变化，这 在图谱中主要表现为蛋白斑点的缺省、增加或位置的改变。相比正常组，仅出现在IPF组 和矽肺组的蛋白斑点有8个，而有31个蛋白斑点仅出现于正常对照组;相比IPF组与矽 肺组，有20个蛋白斑点仅出现在矽肺组，而有25个蛋白质斑点在矽肺组中缺失。 还有部分蛋白质斑点出现了量的差异，这些差异主要表现为斑点染色的深浅改变。统计 两组中出现5倍量差异的点共有98个，其中，相比正常组，有13个点在IPF组及矽肺组 中出现5倍量的增加;而有22个斑点却出现5倍量的减少。相比正常组和IPF组，有38 个蛋白斑点在矽肺组中出现5倍量的增加;而又有25个斑点在矽肺组中出现5倍量的减 少。5.通过网络蛋白质数据库的搜索获得一些初步的蛋白质信息 根据蛋白质斑点的分子量和等电点更多还原

【Abstract】 [Objective]Silicosis is one of the most serious occupational disease in our country. Atpresent, the pathogenetic mechanism of silicosis is unclear. The basic pathological change of silicosis is the lung interstitial fibrosis and the formation of silicotic nodule. Similar to the silicosis, idiopathic pulmonary fibrosis (IPF), which is another lung interstitial disease, is characterized by lung fibrosis. The same character of these two diseases is the persistent inflammation of alveolar and the formation of proinflammatic mediators and profibriotic mediators. So it shows the common origin of the lung fibrosis in these two lung diseases. But compared with IPF and other interstitial lung diseases, silicosis has the remarkable pathological alteration which is the formation of silicotic nodule.The protein compeonent of the BALF derives from the thin layer of epitheliallining fluid of the peripheral airways and alveolar, and may reflect parenchymal disease processes. Because of the complicated pathogenetic mechanisms of lung disease, the research is focused on the proteins and proteomes of BALF nowadays. The mechanism of silicosis is furthering to the molecular and cellular level on the basic research.To explore the various cytokines of lung fibrosis, the solution protein in BALF of normal control, IFF and silicosis were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The 2D maps of the three groups were compared and the differential display protein spots were screened. It lays the foundation for identification of the differential protein by mass spectrum (MS) in the next step. And also it can provide a new way to study the pathogenetic mechanisms of silicosis.[Method]Healthy and male SD rats (weight about 200g) were supplied by the center of laboratory animal in the medical school of zhejiang university. All rats were divided at random into three groups. The health, IFF and silocosis animal model were structured by injecting intratracheally with 1ml 0.9% N.S., 5mg/kg Bloemycin and 1ml suspension of quartz dust (SiO2) at a concentration of 50mg/ml respectively. The bronchoalveolar lavage fluid (BALF) of the three groups was obtained by bronchoalveolar lavage of these animals. The protein quantitation in BALF was measured. The protein were precipitated with TCA(10% final concentration) in an ice bath for 20 min, and subsequently centrifuged at 3500 rpm for 15 min and 4 . The pellet was suspended in ice-cold acetone using a sonicator and centrifuged as described above. The pellet was air-dried for a few minutes and, finally, re-suspended in the sample solution by sonication. A 180 g amount of proteins dissolved in total 300 l Rehydration Buffer (8mol/L urea, 2%CHAPS, 20mmol/L DTT, 0.5%IPG Buffer pH3-10L, Trace Bromophenol blue) were loaded on pH 3-10 linear IPG strips (170mm 3mm 0.5mm) for isoelectric focusing. Second-dimensional separation of the proteins was done on ExcelGel XL 12% SDS-PAGE and detected by silver staining. The BALF 2-DE maps were analyzed by the 2D software PDQuest 7.1. Thedifferential protein spots, which have relationship with lung fibrosis and silicotic nodule, were screened. The data were analyzed by the software SPSS for windows 11.0.[Result]1. Pathology of the rats lung tissueUnder light microscope, it was found that the lung structure of normal control rats was intact and distinct. In the IPF group, the lung structure was also intact but the hyperplasia of the collegen in the lung interstitial was found. And the lung structure was destroyed and the typical silicotic nodule was found in the silicosis group. The cell aggregation and cellular nodule were predominant in this group and the collegen increased in these silicotic nodules. Also the fibroplasia in the lung interstitial tissue was found in these rats.2. Comparing of the protein quantitation in the BALF of the differential groupsThe measured values of the protein quantitations in the BALF of normal, IPF and silicosis were 0.3196 mg/ml, 0.3075 mg/ml and 1.27更多还原