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Research on control mechanisms of growth and differentiation of mouse primordial germ cells and their developmental manipulation

小鼠原始生殖细胞生长分化调控机制及其发育调控研究

基本信息

批准号：
05454655
负责人：
NAKATSUJI Norio
金额：
$ 4.16万
依托单位：
National Institute of Genetics
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

1. Primordial germ cells (PGCs) show proliferation and growth arrest in culture. Such growth pattern could be regulated autonomously or by somatic cells. The mixed culture experiments using PGCs from 8.5 and 11.5 dpc embryos indicated no detectable interaction between the PGCs and somatic cells at the two stages. Clonal culture of PGCs did not reveal any subpopulation of PGCs in terms of growth rate and morphological changes. We conclude that there is an autonomous regulation of growth, which is not strictly timed by the number of cell divisions.2. Leukemia inhibitory factor (LIF) is a cytokine known to influence proliferation and/or survival of mouse PGCs in culture. We have analyzed the functional role of gp130-mediated signaling in PGC growth in vitro. A combination of IL-6 and soluble IL-6 binding subunit (sIL-6R) fully reproduced the LIF action on PGCs. Addition of neutralizing antibody against gp130 in culture remarkably blocked PGC survival. These results suggest the pivotal role of gp130 in PGC development. We have further demonstrated that a combination of LIF with forskolin or retinoic acid caused continuation of the proliferation of PGCs.3. We tested transfection methods of electroporation, liposome-mediated transfection and calcium phosphate (CaPO4) co-precipitation method. When PGCs were transfected with plasmid pSV-LT by CaPO4 co-precipitation method, transient expression of simian virus 40 large-T antigen was detected in an average of 18% of PGCs transfected. Transient expression of adenovirus 2 E1B 19kDa protein in about 15% of PGCs significantly retarded disappearance of PGCs from the culture system and increased the number of rounded PGCs.

1. 原始生殖细胞（PGCs）在培养中表现出增殖和生长停滞。这种生长模式可以由细胞自主调节或由体细胞调节。用8.5 dpc和11.5 dpc胚胎的PGCs进行混合培养实验表明，在这两个阶段，PGCs与体细胞之间没有明显的相互作用。克隆培养在生长速度和形态变化方面未发现PGCs的任何亚群。我们得出的结论是，存在一种生长的自主调节，这种调节并不严格取决于细胞分裂的次数。白血病抑制因子（LIF）是一种已知影响培养小鼠PGCs增殖和/或存活的细胞因子。我们分析了gp130介导的信号在体外PGC生长中的功能作用。IL-6和可溶性IL-6结合亚基（sIL-6R）的结合完全复制了LIF对PGCs的作用。在培养物中加入抗gp130的中和抗体可显著阻断PGC的存活。这些结果表明gp130在PGC的发展中起着关键作用。我们进一步证明，LIF与福斯克林或视黄酸联合使用可导致pgcs的持续增殖。我们测试了电穿孔转染法、脂质体介导转染法和磷酸钙共沉淀法。用CaPO4共沉淀法转染pSV-LT质粒后，平均有18%的PGCs瞬时表达猴病毒40大t抗原。腺病毒2型E1B 19kDa蛋白在约15%的PGCs中短暂表达，显著延缓了PGCs从培养系统中消失，并增加了圆形PGCs的数量。