Bradford C S, Sun L, Barnes D W
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-7305.
Mol Mar Biol Biotechnol. 1994 Apr;3(2):78-86.
We are attempting to develop methods for in vitro culture of zebrafish embryonal stem cells. Primary cultures were initiated from wild-type zebrafish early embryos in basal nutrient medium supplemented with insulin, selenite, leukemia inhibitory factor, trout serum, fetal bovine serum, and trout embryo extract. In this medium, melanocytes appeared on the second day of culture. Basic fibroblast growth factor (bFGF) was mitogenic when cells were plated at low densities. bFGF suppressed melanogenesis is a dose-dependent fashion, with maximal effect at 20 ng/mL. Cultures initiated and maintained with bFGF for 24 hours and then incubated without bFGF for as long as 8 days did not contain pigmented cells. Experiments in which bFGF was added or removed at various times after initiation of cultures indicated that maximum sensitivity to bFGF occurred during the first 12 hours of culture. When wild-type cells from cultures without bFGF were injected into albino blastula-stage embryos, melanocytes subsequently developed in host embryos: no melanocytes appeared when cells from cultures with bFGF were injected into albino hosts.
我们正在尝试开发斑马鱼胚胎干细胞的体外培养方法。原代培养从野生型斑马鱼早期胚胎开始，在添加了胰岛素、亚硒酸盐、白血病抑制因子、鳟鱼血清、胎牛血清和鳟鱼胚胎提取物的基础营养培养基中进行。在这种培养基中，培养第二天出现了黑素细胞。当细胞以低密度接种时，碱性成纤维细胞生长因子（bFGF）具有促有丝分裂作用。bFGF以剂量依赖方式抑制黑素生成，在20 ng/mL时效果最佳。用bFGF启动并维持培养24小时，然后在无bFGF的情况下孵育长达8天的培养物中不含色素细胞。在培养开始后的不同时间添加或去除bFGF的实验表明，培养的前12小时对bFGF的敏感性最高。当将无bFGF培养物中的野生型细胞注射到白化囊胚期胚胎中时，宿主胚胎随后会发育出黑素细胞：当将有bFGF培养物中的细胞注射到白化宿主中时，未出现黑素细胞。
