胎鼠肺细胞的分离纯化及原代培养

Isolation and Purification and Primary Culture of Lung Cells from Fetal Rats

目的 建立一套可靠的胎鼠肺细胞分离、纯化和培养技术 ,用于体外研究肺发育和早产儿肺部疾病。方法采用胰酶和胶原酶消化 19d胎鼠肺组织块 ,经差速离心和反复贴壁法 ,分离、纯化胎鼠肺泡Ⅱ型细胞 (AECⅡ )和肺成纤维细胞 (LF) ,并进行原代培养。用细胞角蛋白 (cytokeratin)和波形蛋白 (vimentin)免疫细胞化学染色和透射电镜对所分离的细胞进行鉴定。结果 该方法所得细胞产量大、纯度高。免疫细胞化学鉴定 ,AECⅡ细胞cytokeratin染色阳性 ,vimentin染色阴性 ,LF则相反。透射电镜观察AECⅡ ,可见细胞内板层小体。结论 该方法是一种可靠的胎鼠肺细胞的分离纯化培养技术 。

Objective To develop a reliable method for isolation, purification and primary culture of type Ⅱ alveolar epithelial cells (AECⅡs) and lung fibroblasts (LFs) from fetal rat lung for studies of lung development and premature lung diseases in vitro . Methods The lung tissue of 19-day fetal rats was digested with trypsin and collagenase. AECⅡs and LFs were isolated and purified in different centrifugal force and different adherence, then cultured. The nature of the cul tures was identified by cytokeratin staining and vimentin staining and electron micrography. Results Excellent yields of highly purified, culturable AECⅡs and LFs could be obtained from 19-day fetal lungs. The expression of cytokerati n in AECⅡs was positive and that of vimentin negative by immunocytochemistry. T hose, however, in LFs were just opposite. Lamellar bodies in purified AECⅡs wer e revealed by ultrastructural examination under electron micrography. Conclusion This technique might be a reliable method for isolat ion and purification and primary culture of alveolar epithelial cells and lung f ibroblasts from fetal lung and could be used for the study of lung development a nd premature lung disease in vitro.