豚鼠肺泡Ⅱ型上皮细胞的分离、纯化与培养

Isolation,purification and culture of Type Ⅱ alveolar epithelial cells from the guinea pig

为了建立豚鼠肺泡Ⅱ型上皮细胞(AECⅡ)分离、纯化和ROCK抑制剂培养的方法,进一步以AECⅡ为模型研究其在抗结核分枝杆菌中的免疫调控作用奠定基础,试验利用混合酶溶液全肺灌注法消化、分离含有豚鼠AECⅡ的混合细胞,经过红细胞裂解液处理与免疫黏附法纯化获得AECⅡ。以3T3细胞为饲养层,并添加Rho激酶抑制剂的方法进行AECⅡ的传代培养。结果表明:混合酶溶液灌注消化法能充分消化肺脏上皮细胞,通过Ig G免疫黏附能有效去除巨噬细胞而纯化AECⅡ,并且在3T3细胞饲养层与ROCK抑制剂培养条件下促进了AECⅡ的体外增殖而抑制其分化。

To establish the method of isolation, purification and culture with Rho- associated protein kinase （ROCK） inhibitor of the type Ⅱ alveolar epithelial cells （ AEC Ⅱ ） from the guinea pig, and further lay a basis for the research on the immune regulation of AEC Ⅱ against Mycobacterium tuberculosis using AEC Ⅱ as a cell model. The mixed cells containing the AEC Ⅱ were digested and isolated by poured the mixed enzyme solution into the intact pulmonary, and then the AEC Ⅱ were obtained through the treatment of erythrocyte lysis buffer and the purification by immune adhering. The AEC Ⅱ were subcuhured with 3T3 feeder cells as a feeder layer and adding with ROCK inhibitor. The result showed that the pulmonary alveolar epithelial cells could be fully digested by perfusion digestion with the mixed enzyme solution. The AEC Ⅱ could be purified through immune adhering with IgG to efficiently remove the maerophages. The in vitro proliferation of AEC Ⅱ were promoted but their differentiation was inhibited under the culture conditions with 333 cell feeder layers and ROCK inhibitor.