博莱霉素诱导胸膜间皮细胞上皮—间充质转换在肺纤维化发生中的作用

发布时间：2018-10-09 11:41

【摘要】：目的：研究博莱霉素是否可诱导胸膜间皮细胞发生上皮-间充质转换（EMT），探讨胸膜间皮细胞发生EMT在特发性肺纤维化发生中的作用。方法：体外培养胸膜间皮细胞，以博莱霉素处理，观察胸膜间皮细胞Ⅰ型胶原表达的变化；Western blot测定胸膜间皮细胞发生上皮-间充质转换时相关表型蛋白的变化；倒置相差显微镜下观察细胞形态的变化；细胞划痕实验检测胸膜间皮细胞的迁移。最后用博莱霉素多次腹腔注射的方法复制C57小鼠肺纤维化模型，Masson三联染色检测肺组织胶原含量、免疫荧光检测胸膜间皮细胞标志物calretinin蛋白表达，从而在体判断胸膜间皮细胞的肺内移行情况。结果：博莱霉素处理胸膜间皮细胞后，Ⅰ型胶原蛋白表达升高，上皮细胞表型蛋白CK-8蛋白表达下降，间充质细胞标志蛋白波形蛋白、α-SMA蛋白表达升高，而与细胞完整性相关的E-钙粘蛋白表达不变。细胞形态在24h发生改变，48h、72h更为明显，由鹅卵石形逐渐拉长向梭形改变。细胞划痕实验提示博莱霉素诱导的细胞迁移明显大于对照组。肺纤维化动物模型中Masson染色结果显示，博莱霉素导致肺内大量纤维化灶形成，以胸膜下为主，伴明显的胸膜增厚，胶原表达含量明显增加；免疫荧光染色显示，肺实质内尤其纤维灶的部位胸膜间皮细胞标志物calretinin蛋白表达明显增多，提示胸膜间皮细胞向肺内移行。结论：博莱霉素可诱导胸膜间皮细胞发生EMT，胸膜间皮细胞EMT在特发性肺纤维化发病机制中可能起重要作用。
[Abstract]:Aim: to investigate whether bleomycin can induce epithelial-mesenchymal transition (EMT),) in pleural mesothelial cells to investigate the role of EMT in the pathogenesis of idiopathic pulmonary fibrosis. Methods: pleural mesothelial cells were cultured in vitro and treated with bleomycin to observe the changes of type I collagen expression in pleural mesothelial cells. The changes of phenotypic proteins associated with epithelial-mesenchymal transition in pleural mesothelial cells were determined by Western blot. Cell morphology was observed under inverted phase contrast microscope and migration of pleural mesothelial cells was detected by cell scratch assay. The pulmonary tissue collagen content was detected by Masson triple staining and the expression of calretinin protein in pleural mesothelial cells was detected by immunofluorescence. Thus the pulmonary migration of pleural mesothelial cells was judged in vivo. Results: after treated with bleomycin, the expression of type I collagen protein increased, the expression of phenotypic protein CK-8 protein decreased, and that of mesenchymal cell marker protein vimentin and 伪 -SMA protein increased. The expression of E-cadherin associated with cell integrity remained unchanged. The change of cell morphology was more obvious at 48h and 72h, from cobblestone to fusiform. Cell scratch test showed that bleomycin induced cell migration was significantly higher than that of control group. The results of Masson staining in pulmonary fibrosis animal model showed that bleomycin caused a large number of pulmonary fibrosis foci, mainly in the subpleural area, accompanied by obvious pleural thickening, collagen expression increased significantly, immunofluorescence staining showed that, The expression of calretinin protein in the pleural mesothelial cells, especially in the fibrous foci, was significantly increased, indicating the migration of pleural mesothelial cells to the lung. Conclusion: bleomycin can induce the development of EMT, pleural mesothelial cells and EMT may play an important role in the pathogenesis of idiopathic pulmonary fibrosis.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563

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