大鼠Ⅰ型肺泡上皮细胞的分离、培养及生物学特性研究  

Isolation, culture and biological characteristics of alveolar epithelial type Ⅰ cells in rats

作　　者：李勇[1] 杜娟[1] 余静[1] 王海燕[1] 岳彩黎[1] 曾灵[1] 蒋建新[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第四研究室,创伤,烧伤与复合伤国家重点实验室,重庆400042

出　　处：《解放军医学杂志》2014年第5期374-378,共5页Medical Journal of Chinese People's Liberation Army

基　　金：国家重点基础研究发展计划(973计划)项目(2012CB518104)~~

摘　　要：目的改良细胞分离方法,得到高纯度的原代Ⅰ型肺泡上皮细胞(ATⅠ)并进行培养,初步观察其生物学特性。方法采用SPF级SD大鼠,通过酶消化法、IgG贴壁法和免疫磁珠法获得高纯度ATⅠ并进行培养,采用台盼蓝染色计算细胞活力,免疫荧光DAPI法鉴定细胞纯度和表型,倒置显微镜观察细胞生长规律,计数法绘制细胞生长曲线。结果每只体重120g左右的SD大鼠可获得(2.1±0.5)×106个ATⅠ,细胞活力为93.8%±1.6%,细胞纯度为91.0%±0.6%。培养3d后细胞完全黏附、伸展,培养7d后汇合形成单层扁平状。免疫荧光检测显示,培养的原代细胞水通道蛋白5(AQP5)和小窝蛋白1(Cav-1)呈阳性表达,表面活性蛋白C前体(pro-SPC)呈阴性表达。获取的ATⅠ可在体外增殖,生长曲线显示原代ATⅠ在对数增长期的倍增时间约为66h。结论改良细胞分离方法可以得到高纯度的ATⅠ,培养后细胞生长良好,可为研究以ATⅠ损伤为特征的肺部疾病提供可靠的体外模型。Objective To obtain and culture high-purity primary alveolar type Ⅰ cells （ATⅠs） by modified isolation method, and observe its biological characteristics, so as to provide a better in vitro model for the study of lung diseases characterized by injury of ATⅠ. Methods High-purity ATⅠs were isolated from specific-pathogen-free （SPF） SD rats by neutral protease digestion, IgG adherent and magnetic sorting methods, and they were then cultured. The cell viability was calculated by trypan blue staining, the cell purity and phenotype were identified by immunofluorescence staining. The cells were observed by inverted microscope and the growth curve was drawn. Results （2.1±0.5）×106 ATⅠs could be harvested from a rat with a body weight of about 120-g, and the viability was 93.8%±1.6%, with a purity of 91.0%±0.6%. After cultured for 3 days, the cells began to spread; by the day 7, the cells formed confluent monolayer. Immunofluorescence staining showed the cultured ATⅠs were positive for aquaporin-5 and caveolin-1, and negative for surfactant protein C precursor （pro-SPC）. The obtained ATⅠs could proliferate in vitro, and the population doubling time as estimated from the logarithmic phase of the growth curve was about 66h. Conclusion High-purity ATⅠs could be obtained by the improved method for cell isolation, and the cells grew well after culture, providing a good in vitro model for the study of lung diseases characterized by ATⅠs damage.

关 键 词：肺泡上皮细胞 Ⅰ型 细胞分离 细胞培养技术 

分 类 号：R135.2[医药卫生—劳动卫生]