TRH REGULATION/BIOSYNTHESIS AND PARAVENTRICULAR NUCLEUS

TRH 调节/生物合成和室旁核

• 批准号: 2139942
• 负责人: RONALD Michael LECHAN
• 金额: $ 24.77万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139942
• 关键词: antisense nucleic acid; cerebrospinal fluid; corticotropin releasing factor; cytokine; fasting; gene induction /repression; glucocorticoids; hormone receptor; hormone regulation /control mechanism; hypothyroidism; immunocytochemistry; inflammation; laboratory rat; neuropeptide Y; neuropharmacology; nucleic acid sequence; oligonucleotides; paraventricular nucleus; peptide hormone biosynthesis; protein sequence; somatostatin; thiophosphate; thyrotropin releasing hormone; transfection; triiodothyronine

The main objectives of these studies are to elucidate the mechanisms of control of the biosynthesis of thyrotropin-releasing hormone (TRH) in a discrete population of neurons in the hypothalamic paraventricular nucleus (PVN) that is critical for the regulation of anterior pituitary TSH secretion and to determine how this regulatory system is perturbed in disease states and disorders characterized by lows circulating thyroid hormone and inappropriately low TSH (sick euthyroid syndrome). It is proposed to accomplish these goals by combining methods of molecular hybridization with classical neuroanatomical techniques and immunocytochemistry at the light and ultrastructral levels. The thyroid hormone receptor (TR)subtypes in hypophysiotropic TRH neurons as well as in neurons in other regions of the brains will be identified by double- labeling immunocytochemistry using specific antiseria to TRalpha1, TRbeta1, TRbeta2, and the TR variant, TRalpha2, respectively and antiserum to the TRH precursor. The role each receptor in mediating feedback effects of thyroid hormone will be further assessed by semiquanitative in situ hybridization histochemistry and computerized image analysis after inhibiting TR expression with specific, antisense, phosphorothioate oligodeoxynucleotides (S-oligos). The efficacy of S-oligos will be validated by in in vitro bioassay systems including in vitro translation and transient transfection assays before injection into the third ventricle or adjacent to the PVN. The possibility that a material with TRbeta2-like immunoreactivity represents a novel thyroid hormone receptor in hypophysiotropic TRH neurons will be addressed by expression cloning to identify its full length cDNA. The subcellular location of TRs and their association with T3 will be identified by electron microscopy and autoradiography with the intent of providing anatomical evidence for a role of TRs as anterogradely transported proteins in neuronal processes. The role of the blood-brain barrier versus the blood-CSF barrier in contributing to feedback regulation of TRH biosynthesis in the PVN will be assessed by semiquanitative in situ hybridization histochemistry and image analysis after inhibiting the movement of systemically administered T4 into the CSF of hypothyroid animals by saturating transthyretin binding sites in the CSF with the flavonoid, EMD 21388. The mechanisms by which the normal feedback effects of thyroid hormone on TRH neurons in the PVN is superseded by conditions that result in the sick euthyroid syndrome, will be studied in fasted animals and following central administration of inflammatory cytokines (Il-1beta, IL-6, TNFalpha). The role of glucocorticoids, neuropeptide-Y, somatostatin, corticotropin-releasing hormone and alternations in TR concentration in mediating these responses will be determined using similar neuroanatomic methods.

这些研究的主要目标是阐明 在A中控制甲状腺蛋白释放激素（TRH）的生物合成 下丘脑旁脑核中神经元的离散群体 （PVN）对于调节前垂体TSH至关重要 分泌并确定该调节系统的干扰 疾病状态和疾病的特征是低循环甲状腺的疾病 激素和不适当的低TSH（病态甲状腺功能综合征）。这是 提议通过结合分子方法来实现这些目标 与经典神经解剖技术的杂交和 在光和超疗法水平上的免疫细胞化学。甲状腺 低生理TRH神经元中的激素受体（TR）亚型以及 在大脑其他地区的神经元中，将通过双重识别 使用特定抗tralpha1的特定抗溶质化对免疫细胞化学进行标记， TRBETA1，TRBETA2和TR变体TRALPHA2和抗血清 到TRH前体。每个受体在中介反馈效应中的作用 甲状腺激素的原位将进一步评估 杂交组织化学和计算机图像分析之后 用特异性，反义，磷酸盐剂抑制TR表达 寡脱氧核苷酸（S-Oligos）。 S-Oligos的功效将是 通过体外生物测定系统的验证，包括体外翻译 并在注射到第三次之前进行瞬态转染测定 心室或毗邻PVN。与 TRBETA2样免疫反应性代表一种新型的甲状腺激素受体 在低生理的TRH神经元中，将通过表达克隆来解决 确定其全长cDNA。 TR的亚细胞位置及其 与T3的关联将通过电子显微镜和 放射自显影是为了提供解剖学证据 在神经元过程中，TR作为同类转运蛋白的作用。 血脑屏障与血液-CSF屏障的作用 促进PVN中TRH生物合成的反馈调节将是 通过半定性原位杂交组织化学和图像评估 抑制系统管理的T4运动后的分析 通过饱和甲状腺素蛋白结合，进入甲状腺功能减退动物的CSF 带有类黄酮的CSF的站点，EMD 21388。 甲状腺激素对PVN中TRH神经元的正常反馈作用 被导致病人甲利治体综合征的条件所取代 将在禁食动物中进行研究，并在中央给药后 炎性细胞因子（IL-1Beta，IL-6，TNFalpha）。的作用 糖皮质激素，神经肽-Y，生长抑素，皮质激素释放释放 激素和TR浓度的交替介导这些反应 将使用类似的神经解剖学方法确定。