权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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）进入细胞是一个关键，但不太了解的过程。 TH进入细胞的改变可能有助于甲状腺激素代谢的变化，禁食，在重症患者（NTI），甲状腺功能减退症，并作为一个毒品的后果。 TH进入细胞的改变已经被证实是在异常TH血清结合蛋白患者中描述作为一种主要的遗传缺陷，抗TH。这些研究的长期目标是阐明甲状腺进入的机制和调节激素进入人体细胞，并评估其贡献，临床问题。将培养的人肝细胞Hep G2 因为它们保留了正常人类肝脏的许多特性细胞，并允许控制实验条件。将这些细胞与放射性碘标记的T3和T4孵育，未标记的TH和无活性代谢物将建立，如果人肝细胞具有TH的饱和摄取系统，在生理浓度下具有功能性，并对活性物质具有特异性日用不同浓度的游离T3和T4孵育，纯化白蛋白、甲状腺素结合前白蛋白（TBPA），以及甲状腺素结合球蛋白（TBG）将指示这些蛋白质是否促进TH的吸收。用放射性碘孵育细胞标记的白蛋白，TBPA和TBG将决定这些血清蛋白特异性结合于人肝细胞。血清异常者对示踪TH的摄取 TH结合蛋白将评估这些蛋白质是否改变了细胞的功能。细胞摄取 TH与呋塞米和游离脂肪酸的孵育酸将决定这些假定的TH抑制剂是否与NTI中的血清蛋白结合也干扰细胞进入理论产率用细胞阻滞剂处理Hep G2细胞 ATP的产生将表明甲状腺的摄取是否激素进入人体细胞是能量依赖的。预处理与抑制内吞作用的药物联合使用，这一过程在人细胞摄取TH中很重要。将从Hep G2细胞获得的膜制备物结合不同浓度的标记和未标记T3 和T4来检测特定的膜受体。孵育用伊泊酸、胺碘酮和普萘洛尔标记TH，如果它们通过竞争摄取而改变TH的细胞进入，抑制脱碘或降低细胞ATP含量。的甲状腺功能低下对Hep G2细胞长期作用的影响血清和血清与过量TH将确定是否甲状腺激素状态可改变TH进入人体细胞。