TRANSPORT AND ACTION OF THYROID HORMONES

这项研究计划的长期目标是了解 甲状腺激素作用的分子基础。此应用程序 解决了甲状腺激素如何促进 细胞生长。 甲状腺激素是GH4C1生长所必需的 大鼠垂体细胞，如果细胞以低密度培养 中等水平的甲状腺激素缺乏。我们已经证明了甲状腺 激素通过刺激细胞来促进GH4C1细胞分裂 分泌一种自分泌生长因子。这个增长因素完全是 取代了对甲状腺激素的需求。我们还有 分离出一种能够正常生长的变异细胞系 没有添加任何甲状腺激素，但甲状腺功能正常 荷尔蒙反应性。 我们计划提纯和描述甲状腺的活动。 激素诱导的垂体细胞分泌的生长因子。我们 将首先优化条件，最大限度地分泌 活动。然后我们将进行基本的生化 一个或多个增长因素的特征，这些因素将提供 分子大小和结构的指示。增长因素 将通过传统的生化技术进行提纯 促进生长或刺激掺入放射性物质 胸腺嘧啶核苷或放射性氨基酸摄取作为一种生物测定 净化。同时，我们将尝试用放射性标签 生长因子和鉴定候选多肽。这将是 通过使用突变体能够在没有 甲状腺激素；这条线路应该分泌生长因子或 没有甲状腺激素，而父母系应该分泌 只有在荷尔蒙存在的情况下才有生长因子。如果推定的 生长因子被确定，它将用于生成 抗体。 纯化的甲状腺激素诱导生长因子将用于 研究其在GH4C1细胞株中的作用机制。我们会 确定生长因子是否具有任何固有的类甲状腺激素 活动，超越生长刺激，并研究细胞 在静止细胞中加入生长因子后的变化。

The long range goal of this research program is to understand the molecular basis of thyroid hormone action. This application addresses the specific question of how the thyroid hormones promote cell growth. Thyroid hormone is an absolute requirement for the growth of GH4C1 rat pituitary cells if the cells are plated at low density in medium depleted of thyroid hormones. We have shown that thyroid hormones promote GH4C1 cell division by stimulating the cells to secrete an autocrine growth factor. This growth factor totally replaces the requirement for thyroid hormone. We have also isolated a variant line of cells that is able to grow normally without any added thyroid hormone but has otherwise normal thyroid hormone responsivity. We plan to purify and characterize the actions of the thyroid hormone induced growth factor secreted by pituitary cells. We will first optimize the conditions for maximal secretion of the activity. We will then carry out basic biochemical characterization of the growth factor, or factors, which will give an indication of molecular size and structure. The growth factor will be purified by conventional biochemical techniques using either growth promotion or stimulated incorporation of radioactive thymidine or uptake of radioactive amino acids as a bioassay during purification. In parallel, we will attempt to radioactively label the growth factor and identify candidate peptides. This will be facilitated by using the mutant able to grow in the absence of thyroid hormones; this line should secrete growth factor with or without thyroid hormones, whereas the parental line should secrete growth factor only in the presence of the hormone. If a putative growth factor is identified it will be used for the generation of antibodies. Purified thyroid hormone induced growth factor will be used to study the mechanism of action in the GH4C1 cell line. We will determine whether the growth factor has any intrinsic thyromimetic activities, beyond growth stimulation, and study the cellular changes following addition of the growth factor to quiescent cells.