摘要
【目的】建立一套可靠的早产鼠肺泡Ⅱ型上皮细胞（AECⅡ）分离、纯化和培养技术，用于体外研究早产儿肺部疾病。【方法】采用胰酶和胶原酶消化胎龄20d早产鼠肺组织块，经差速离心和反复贴壁法，分离、纯化早产鼠AECⅡ，并进行原代培养。用细胞角化蛋白（cytokeratin）进行二氨基联苯胺（DAB）及荧光免疫细胞化学显色，和透射电镜对所分离的细胞进行鉴定。【结果】AECⅡ体外培养24～48h，增殖代谢旺盛，进入对数生长期，生长状态最佳。cytokeratin在DAB免疫细胞化学及荧光免疫细胞化学中均有表达。透射电镜观察可见细胞内板层小体。【结论】该方法所得细胞产量大、纯度高，是一种可靠的早产鼠AECⅡ的分离纯化培养技术，对体外研究早产儿肺部疾病如高氧肺损伤等有重要意义。
[Objective] To develop a reliable method for isolation, purification and primary culture of type Ⅱ alveolar epithelial cells（AEC Ⅱ s） from premature rat lung for studies of premature lung diseases in vitro. [Methods] The lung tissue of 20day fetal rats was digested with trypsin and collagenase. AEC Ⅱ s were isolated and purified in different centrifugal force and different adherences, then cultured. The nature of the cultures was identified by immunocytochemieal staining of diaminobenzidine（DAB） and fluorescent immuocytochemistry with cytokeratin, and electron micrography. [Results]This method achieved highly purified and excellent yields . Cuhurable AEC Ⅱ s could be obtained from 20-day fetal lungs. When AEC Ⅱ s were cultured for 24-48 hours, their proliferation and metabolism were most productive, and entered logarithmic phase. The expression of cytokeratin in AEC Ⅱs was all positive by immunocytochemical identification of DAB and identification of fluorescence immunocytochemistry. Lamellar bodies in purified AEC Ⅱ s were revealed by uhrastructural examination under electron mierography. [Conclusion] This technique might be a reliable method for isolation, purification, and primary culture of AECⅡ s from premature rat lung. It plays an important role in lung disease of premature infants, such as hyperoxic lung injury.
出处
《医学临床研究》
CAS
2009年第3期377-380,共4页
Journal of Clinical Research
基金
湖南省教育厅科研基金资助项目（07C572）
关键词
肺泡/细胞学
上皮细胞
pulmonary alveoli/CY
epithelial cells
