小鼠实验性肺纤维化上皮细胞-间充质细胞转变及骨桥蛋白的表达

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。

EXPERIMENTAL STUDY ON EPITHELIAL-MESENCHYMAL TRANSITION AND OSTEOPONTIN IN MICE BLEOMYCIN-INDUCED PULMONARY FIBROSIS

Objective To confirm epithelial mesenchymal transition(EMT) and the expression of osteopontin (OPN) in various periods of mice BLM induced pulmonary fibrosis by semi quantitative analysis and pathomorphological evaluation. Methods sixty healthy adult male C57BL/6 mice were randomly divided into the NS-Control group and the BLM injured group，which were treated with 50μl NS and 50μl BLM solution by oropharyngeal aspiration respectively. Left lung tissues were taken from all the mice and paraffin imbedding and section were performed separately on days 3,7,14,21,28 after the treatment. The histopathological changes and total lung collagen content of mice were determined by hematoxylin eosin staining and sirius red staining. The expressions of OPN,SP-C,E-Cadherin、FSP1 and vimentin were detected by immunohistochemical staining. Integrated absorbance(IA)of stained sections was measured by Image Pro Plus 6.0, and then the statistical analysis was done. Results The morphological changes of mice lung tissue in various periods were demonstrated by HE staining. Total lung collagen content of BLM group increased during the pulmonary fibrosis progress by picrosirius polarization staining (P<0.05) In immunohistochemical staining, the expression of OPN in the BLM group was more significant than that in the NS group, peaking on day 21-28; lots of SP-C positive typeⅡalveolar cell of normal size were observed in the NS group, and a progressive amount of positive cells in the fibrous focal zone with bigger cell body was seen in the BLM group; a few of E- Cadherin positive alveolar epithelial cells could be seen in the NS treated lung and 14-day and 21-day BLM injured