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目的从形态学上证实小鼠实验性肺纤维化上皮细胞-间充质细胞转变(EMT)及骨桥蛋白(OPN)的表达变化。方法将60只健康雄性C57BL/6小鼠随机分为生理盐水对照组和博来霉素(BLM)模型组,采用口咽抽吸法经咽部注入气管50μl博来霉素溶液,对照组注入50μl生理盐水。分别在造模后第3d、7d、14d、21d及28d 5个时间点处死对照组及模型组小鼠。取小鼠左肺制备石蜡切片,行苏木素-伊红(HE)染色及天狼猩红染色观察肺组织形态变化及胶原增生情况;行表面活性物质相关蛋白-C(SP-C)、上皮型钙黏蛋白(E-Cadherin)、波形蛋白(vimentin)、成纤维细胞特异蛋白1(FSP1)和骨桥蛋白(OPN)免疫组织化学染色,观察肺纤维化上皮细胞-间充质细胞转变(EMT)形态与OPN表达,利用图像分析软件Image-Pro Plus 6.0(IPP6.0)测量切片积分吸光度(IA),并进行统计分析。结果HE及天狼猩红染色显示出小鼠肺组织正常结构和纤维化形态变化,并且模型组小鼠肺组织的胶原生成量随时间推移而增加(P<0.05)。免疫组织化学结果显示,模型组小鼠肺OPN表达量随病程进展持续升高,在纤维化期达到峰值,而对照组表达量很少(P<0.05);对照组SP-C在Ⅱ型肺泡细胞恒定表达,模型组SP-C持续增加,且胞体增大;对照组E-Cadherin表达量多,而模型组在肺实变部位几乎不表达,但肺实变部位产生波形蛋白及FSP1,这些蛋白提示肺纤维化存在EMT。结论BLM诱导性肺纤维化中存在EMT,且与显著增加的OPN有潜在关系。
Objective To confirm the morphological changes of the experimental pulmonary fibrosis epithelial cells - mesenchymal transition (EMT) and osteopontin (OPN) expression in mice. Methods Sixty healthy male C57BL / 6 mice were randomly divided into normal saline control group and bleomycin (BLM) model group. The rats in the control group were injected with 50μl bleomycin solution through the oropharynx in the trachea, Physiological saline. The mice in the control group and the model group were sacrificed at the 5th, 7th, 14th, 21st, and 28th day after modeling respectively. The left lung of the mice was used to prepare paraffin sections. The lung tissue morphology and collagen hyperplasia were observed under hematoxylin-eosin (HE) staining and Sirius red staining. The expressions of surfactant-associated protein C (SP-C), epithelial calcium E-cadherin, vimentin, fibroblast-specific protein 1 (FSP1) and osteopontin (OPN) were detected by immunohistochemical staining to observe the changes of epithelial cells-mesenchymal transition Morphology and OPN expression were measured by using Image-Pro Plus 6.0 (IPP6.0). The integrals of absorbance (IA) were measured and statistically analyzed. Results HE and Sirius red staining showed the normal lung structure and fibrosis in mice. The collagen production in lungs of model mice increased with time (P <0.05). The results of immunohistochemistry showed that the expression of OPN in the lung of model group continued to increase with the progress of the disease and peaked in the fibrosis stage, while the expression of OPN in the control group was very low (P <0.05) The expression of E-Cadherin in the control group was more than that in the model group, while the model group was almost not expressed in the lung consolidation site, but the vimentin and FSP1 were produced in the solidified area of the lung Protein suggests EMT exists in pulmonary fibrosis. Conclusions EMT exists in BLM-induced pulmonary fibrosis and is potentially associated with significantly increased OPN.