大鼠肺泡Ⅱ型上皮细胞的体外培养及内毒素攻击模型的建立  

Establishment of an endotoxin-incluced acute lung injury model on the rat alveolar type Ⅱ cells

作　　者：梅虹霞[1] 金胜威[1] 连庆泉[1] 

机构地区：[1]温州医学院附属第二医院麻醉科,325000

出　　处：《浙江医学》2010年第3期349-352,共4页Zhejiang Medical Journal

基　　金：国家自然科学基金资助项目（30772088）,浙江省卫生高层次创新人才培养工程项目

摘　　要：目的探讨大鼠肺泡Ⅱ型上皮细胞（ATⅡ）体外培养的方法及建立脂多糖（LPS）攻击ATⅡ的模型模拟内毒素性肺损伤。方法分离、纯化、鉴定得到大鼠ATⅡ，分别用01、1、10μg／ml的LPS刺激ATⅡ，并利用细胞增殖／毒性检测试剂在刺激2、4、6h后分别检测ATⅡ的抑制率，从而探索内毒素性肺损伤细胞模型中最适的LPS浓度和刺激时间。结果用碱性磷酸酶染色及电镜观察进行细胞纯度判断，未用差异贴壁去除成纤维细胞和IgG免疫黏附去除巨噬细胞纯化前，纯度达（70．4±4．8）％，纯化后可提高至（90．2±3．4）％；而用LPS干预ATⅡ，ATⅡ出现明显的细胞抑制。其中刺激浓度为1μg／ml时与其他3个实验组相比，细胞毒性反应最大（P〈0．01），且在各个浓度的不同刺激时间里，4h时，细胞毒性反应最大（P〈0．01）。结论细胞分离后采用差异贴壁法和免疫黏附法进行纯化可使细胞纯度提高18％左右l细胞培养时接种密度达到1X107／ml以上有利于细胞的贴壁和生长，而用1μg／ml LPS刺激体外培养的大鼠ATⅡ4h能建立较为合理的内毒素性肺损伤细胞模型。Objective To establish a model of endotoxin-induced acute lung injury on the cultured type II alveolar cells （AT Ⅱ ） of rats. Methods TypeⅡ rat alveolar cells were isolated and purified, and the changes in cellular ultrastructure were studied by electron microscope. AT II cells were treated with 0.1, 1 and 10 p g/ml lipopolysaccharide （LPS）, and the inhibition rate of ATII cells after 2, 4 and 6h of treatment was measured by Cell Counting Kit-8 （CCK-8） method. Results The purity of ATII cells before purification was 70.4 ± 4.8% as identified with AKP stain, that was increased to 90.2 ± 3.4% after purification. LPS induced necrosis and apoptosis of cultured AT II cells after exposed to different doses of LPS; 1 μg/ml LPS resulted in the maximal cytotoxicity to AT II cells （P〈0.01）; there was more severe cell injury after 4h of exposure than 2h and 6h （P〈0.01）. Conclusion The rat type Ⅱ alveolar cells have been purified and an endotoxin-induced lung injury model with exposure to lipopolysaccaride has been successfully established in the study.

关 键 词：肺泡Ⅱ型上皮细胞 细胞分离 内毒素性肺损伤 AT Ⅱ细胞增殖/毒性检测 

分 类 号：R563[医药卫生—呼吸系统]