肺成纤维细胞分离.doc

【摘要】 目的： 建立一套可靠的鼠肺细胞分离、纯化和培养技术,用于体外研究间质性肺病的发病机制及治疗方法。方法： 采用胰酶消化出生后2 d SD大鼠肺组织块及SD大鼠肺泡灌洗液, 经差时贴壁法, 分离、纯化红皮鼠肺成纤维细胞(lung fibroblast, LF)和SD大鼠肺泡巨噬细胞（pulmonary alveolar macrophage,AM）, 并进行原代培养。用波形蛋白(Vimentin)、 CD14细胞免疫荧光染色、蛋白质印迹和电镜对所分离的细胞进行鉴定。结果： 所得细胞产量大、纯度高。细胞免疫荧光染色LF细胞波形蛋白染色阳性而CD14染色阴性；AM细胞则相反。扫描电镜观察可见肺成纤维细胞有微绒毛，细胞间连接成网状，蛋白质印迹结果显示波形蛋白表达。结论： 该方法是一种可靠的红皮鼠肺成纤维细胞及大鼠肺巨噬细胞的分离纯化培养技术, 可用于间质性肺病（interstitial lung disease, ILD）的发病机制及治疗方法的体外研究。 【关键词】 大鼠; 　 肺成纤维细胞; 　 巨噬细胞; 　 原代细胞培养 ［基金项目］ 江苏省高校自然科学研究计划项目（03KJD320076）;镇江市社会发展科技计划项目（SH2004043） 　　　　Isolation and purification and primary culture of lung cells from rats 　　YAN Yu-lan, LIU Yang, BU Xue-feng, ZHANG Zhi-jian, ZHENG Jin-xu 　　(Department of Respiratory Medicine, the Affiliated People′s Hospital of Jiangsu University, Zhenjiang Jiangsu 212002; School of Medicine， Jiangsu University, Zhenjiang Jiangsu 212013,China) 　　［Abstract］ Objective: To develope a reliable method for isolation, purification and primary culture of lung fibroblast(LF) and pulmonary alveolar macrophage(AM) from rat for studies on pathogenesis and treatment of interstitial lung disease in vitro. Methods： The lung tissue of 2 days old immature rats was digested with trypsin.LFs and AMs were isolated and purified, then cultured. The nature of the cultures was identified by CD14 staining, vimentin staining, electron micrography and Western blot. Results： Excellent yields of highly purified, culturable AMs and LFs could be obtained from adult rats and 2 day babyish rat lungs,respectively. The expression of CD14 in AMs was positive and that of vimentin negative by immunofluorescence chemistry. Those, however, in LFs were just opposite. Ramified stracture between purified LFs were observed by ultrastructural examination under electron micrography.Vimentin in purified LFs was revealed by Western blot. Conclusion： This technique might be a reliable method for isolation and purification and primary culture of pulmonary alveolar macrophages and lung fib