新生大鼠肺成纤维细胞的原代培养改良法及细胞鉴定

探讨新生大鼠肺成纤维细胞原代培养的改良方法及细胞鉴定。用胰酶消化组织块结合的方法提取新生大鼠肺成纤维细胞,并纯化细胞,对肺成纤维细胞进行形态学观察,用HE染色及免疫组化染色法对细胞进行鉴定,并用MTT法测定细胞生长曲线。倒置相差显微镜下观察选用改良法获得的细胞,3 d后可见组织块周边有少许细胞,5 d后组织块周围有大量细胞爬出,生长迅速,10 d接近融合。经改良后的方法纯化细胞,细胞活性状态较好的为3~5代,5代以后的细胞增殖能力下降。对第3代肺成纤维细胞进行HE染色,镜下可见形态典型的成纤维细胞,免疫组化结果显示波形蛋白(Vi-mentin)阳性表达,细胞角蛋白(cytokeratin)阴性表达。MTT法检测第3代细胞于3~5 d处于对数生长期。胰酶消化组织块结合法是一种可靠快速的肺成纤维细胞分离纯化的培养方法,使用这种方法可得到具有典型形态特征且活性较好的肺成纤维细胞,初学者容易掌握。

Improved Separation Method and Identification of Primary Fibroblasts from New Born Sprague Dawley Rats

To study the improved separation method and identification of primary fibroblasts from new born Sprague Dawley Rats,we used the methods of trypsin digestion,tissue adherent and the improved separation methods to separate the lung fibroblasts. Then,the primary fibroblasts were purified by two different methods and observed with inverted phase contrast-microscope. At the same time,hematoxylin eosin staining and vimentin immunoh-istochemical staining were conducted to identify fibroblasts. Cell growth curve was evaluated using a color-metric assay with 3-4,5-dimethylthiazol-2-yl]-2,5-diphenyltenyletrazolium bromide(MTT). After 3 days,we could observe the fibroblasts growing out of the tissue and growing rapidly in the fifth day and growing to confluence in the tenth day by using the improved separation method. The cell groups became only fibroblasts purified by improved method. Cells with passage 2 to 5 group were in well viability and it showed a decreased proliferation from passage 6. The typical morphology of the passage 2 cells could be seen under the electron microscope and immunocytochemical staining was positive with vimentin. The growth curve showed the lung fibroblasts in the second and fifth days were in the logarithmic growth phase. The improved separation method was a feasible,simple and rapid method to get fibroblasts of typical characteristics and good viability.