CELLULAR UPTAKE OF THYROID HORMONE BY HUMAN HEPATOCYTES

人肝细胞对甲状腺激素的细胞摄取

基本信息

批准号：
3463406
负责人：
DAVID H SARNE
金额：
$ 9.27万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-08-07 至 1991-07-31
项目状态：
已结题

项目摘要

The entry of thyroid hormone (TH) into cells is a critical, but poorly understood process. Alterations of TH entry into cells may contribute to changes in thyroid hormone metabolism during fasting, in severely ill patients (NTI), in hypothyroidism, and as a consequence of drugs. Altered entry of TH into cells has been described in patients with abnormal TH serum binding proteins and as a primary inherited defect producing generalized resistance to TH. The long term goal of these studies is to delineate the mechanisms and regulation of the entry of thyroid hormone into human cells and to evaluate its contributions to clinical problems. Cultured human hepatocytes, Hep G2, will be utilized as they retain many properties of normal human liver cells and allow experimental conditions to be controlled. Incubation of these cells with radioiodine labeled T3 and T4 with unlabeled TH and inactive metabolites will establish if human liver cells have saturable uptake systems for TH that are functional at physiologic concentrations, and specific for active TH. Incubation with varying concentrations of free T3 and T4 and purified albumin, thyroxine binding prealbumin (TBPA), and thyroxine binding globulin (TBG) will indicate if these proteins facilitate uptake of TH. Incubation of cells with radioiodine labeled albumin, TBPA, and TBG will determine whether these serum proteins are specifically bound to human liver cells. Uptake of tracer TH from serum of subjects with abnormal serum TH binding proteins will evaluate whether these proteins alter the cellular uptake. Incubation of TH with furosemide and free fatty acids will determine whether these postulated inhibitors of TH binding to serum proteins in NTI also interfere with cellular entry of TH. Treatment of Hep G2 cells with drugs which block cellular ATP generation will indicate whether the uptake of thyroid hormone into human cells is energy dependent. Pre-treatment with drugs which inhibit endocytosis will indicate whether this process is important in the uptake of TH by human cells. Membrane preparations obtained from the Hep G2 cells will be combined with varying concentrations of labeled and unlabeled T3 and T4 to detect specific membrane receptors. Incubation of labeled TH with ipodate, amiodarone and propranolol will evaluate if they alter cellular entry of TH by competing for uptake, inhibiting deiodination, or decreasing cellular ATP content. The effect of long term treatment of Hep G2 cells with hypothyroid serum and serum with excess TH will establish whether thyroid hormone status may alter the entry of TH into human cells.

甲状腺激素（Th）进入细胞是关键的，但理解的过程不足。进入细胞的变化可能促进甲状腺激素代谢的变化在严重患者（NTI）中禁食，甲状腺功能减退症，作为一个药物的结果。 TH进入细胞的变化已有在异常TH血清结合蛋白的患者中描述作为主要的遗传缺陷，产生了广义抵抗Th。这些研究的长期目标是描述甲状腺进入的机制和调节激素进入人类细胞并评估其对临床问题。培养的人肝细胞Hep G2将是使用，因为它们保留了正常人肝的许多特性细胞并允许控制实验条件。将这些细胞与标记T3和T4标记的放射性碘一起孵育未标记的TH和非活性代谢物将确定是否人肝细胞具有可饱和的吸收系统在生理浓度下的功能，特定于主动 Th。与自由T3和T4的不同浓度孵育，纯化的白蛋白，甲状腺素结合prealbumin（TBPA）和甲状腺素结合球蛋白（TBG）将指示这些蛋白是否是否促进Th的吸收。将细胞与放射性碘一起孵育标记为白蛋白，TBPA和TBG将确定这些是否是否血清蛋白专门与人肝细胞结合。从异常血清的血清中吸收示踪剂Th TH结合蛋白将评估这些蛋白是否改变细胞摄取。与速尿和游离脂肪一起孵育酸将确定这些假定的抑制剂是否 NTI中与血清蛋白的结合也干扰了细胞进入 th。用阻断细胞的药物治疗HEP G2细胞 ATP生成将指示甲状腺的吸收激素进入人类细胞是能量依赖性的。预处理抑制内吞作用的药物将表明这是否过程在人类细胞对TH的吸收中很重要。从HEP G2细胞获得的膜制剂将是与标记和未标记的T3结合不同的浓度和T4检测特定的膜受体。孵化用iPodate标记为Th，胺碘酮和普萘洛尔将评估如果他们通过竞争吸收来改变TH的细胞进入，抑制去牙，或降低细胞ATP含量。这长期治疗HEP G2细胞用甲状腺功能减退的影响血清和血清多余的血清将确定甲状腺是否激素状态可能会改变TH进入人类细胞。