Kumar R K, O'Grady R, Li W, Smith L W, Rhodes G C
School of Pathology, University of New South Wales, Kensington, Australia.
Exp Cell Res. 1991 Apr;193(2):398-404. doi: 10.1016/0014-4827(91)90112-8.
We report a completely serum-free system for primary culture of fibroblasts from explants of adult mouse lung tissue which permits bioassays for cytokine activity to be performed using unselected populations of cells at low passage number, without interference by serum binding proteins or interacting growth factors. Cultures were established on collagen-coated surfaces in medium MCDB 201 containing albumin, transferrin, epidermal growth factor, lipids, prostaglandin E1, vitamin E, and reducing agents. The cells were morphologically and ultrastructurally typical of fibroblasts in culture and demonstrated expression of vimentin and induction of expression of desmin in culture. Proliferation of the cells was reproducible between different primary cultures and was growth factor dependent. Both cycling and growth-arrested cells exhibited increased DNA synthesis when stimulated with epidermal growth factor, platelet-derived growth factor, or basic fibroblast growth factor, which functioned as complete mitogens, but did not respond to insulin, tumor necrosis factor or interleukin-1 beta. Maximal induction of DNA synthesis by epidermal growth factor required the continued presence of the mitogen in the culture medium. These results cannot be satisfactorily explained by the competence-progression model of responses to mitogenic stimuli but support and extend the findings of other studies using diploid fibroblasts.
我们报道了一种用于从成年小鼠肺组织外植体中进行成纤维细胞原代培养的完全无血清系统，该系统允许在低传代次数下使用未选择的细胞群体进行细胞因子活性的生物测定，而不受血清结合蛋白或相互作用生长因子的干扰。在含有白蛋白、转铁蛋白、表皮生长因子、脂质、前列腺素E1、维生素E和还原剂的MCDB 201培养基中，在胶原包被的表面上建立培养物。这些细胞在形态和超微结构上是培养中成纤维细胞的典型特征，并在培养中显示波形蛋白的表达和结蛋白表达的诱导。不同原代培养物之间细胞的增殖是可重复的，并且依赖于生长因子。当用表皮生长因子、血小板衍生生长因子或碱性成纤维细胞生长因子刺激时，处于增殖周期和生长停滞的细胞均表现出DNA合成增加，这些因子起完全有丝分裂原的作用，但对胰岛素、肿瘤坏死因子或白细胞介素-1β无反应。表皮生长因子对DNA合成的最大诱导需要有丝分裂原持续存在于培养基中。这些结果不能用对有丝分裂刺激的感受态-进展模型得到满意解释，但支持并扩展了其他使用二倍体成纤维细胞的研究结果。
