AN APPLICATION OF IN SITU HYBRIDIZATION AND ANTISENSE OLIGONUCLEOTIDE METHOD TO VISUALIZE AND MANIPULATE GENE EXPRESSION OF STEROID HORMONE RECEPTOR PROTEINS IN THE NERVOUS SYSTEM

应用原位杂交和反义寡核苷酸方法可视化和操纵神经系统中类固醇激素受体蛋白的基因表达

基本信息

批准号：
05454674
负责人：
KAWATA Mitsuhiro
金额：
$ 4.16万
依托单位：
KYOTO PREFECTURAL UNIVERSITY OF MEDICINE
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 1994
项目状态：
已结题

项目摘要

Chromaffin cells were found to contain glucocorticoid receptor-immunoreactivity in their nucleus. When dexamethasone was added into the culture medium, the extension of the processes, intensity of immunoreactivity of MAP-2 were significantly diminished. These results indicated that the differentiation of chromaffin cells to neurons is determined, at least in part, by humoral factor of glucocorticoid through its receptor. The addition of antisense oligos for glucocorticoid receptor cleary caused the significant extension of processes of chromaffin cells, indicating that the protein synthesis of steroid receptor was blocked by the antisense inhibitory action to its mRNA.In the sexually dimorphic area of the hypothalamus, immunocytochemical studies show that estrogen induces a significant changes of numbers of immunoreactive never fibers containing neurotensin, calcitonin-gene related peptide (CGRP), Met-enkephalin, cholecystokinin-8 (CCK-8) and dopamine, indicating that axonal sprouting … More of neurons containing these chemical messengers occur in response to estrogen. The relationship between estrogen-receptor and gene expression of these biochemical messengers in concomitant with the process formation or elongation has been discussed, particularly in the context of male and female differences. Estrogen-receptor was found to localize in the cell nucleus by immunocytochemistry in conjunction with the use of estrogen-receptor antibody. Colchicine treatment blocked immunpositive reaction in the cell nucleus, suggesting that estrogen-receptor protein was synthesized in the cytoplasm followed by the transport to the nucleus through nuclear pore in association with microtubules. Monolayr cultures from male and female rat hypothalami were developed and the differences of neurite extension was investigated. In this point, it should be noted that developmental and estrogen-induced changes in mRNAs coding specific proteins for the cytoskeleton, including tubulin-like molecules, have been indentified in the hypothalamic preoptic area. Less

发现嗜铬细胞的细胞核中含有糖皮质激素受体免疫反应性，当培养基中添加地塞米松时，MAP-2的突起的延伸和免疫反应性的强度显着减弱。这些结果表明嗜铬细胞向细胞分化。神经元至少部分地由糖皮质激素通过其受体的体液因子决定。添加糖皮质激素受体的反义寡核苷酸显然导致了显着的延伸。嗜铬细胞的过程，表明类固醇受体的蛋白质合成被对其 mRNA 的反义抑制作用所阻断。在下丘脑的性二态性区域，免疫细胞化学研究表明，雌激素会引起免疫反应性数量的显着变化。纤维含有神经降压素、降钙素基因相关肽（CGRP）、甲硫氨酸脑啡肽、胆囊收缩素-8（CCK-8）和多巴胺，表明轴突含有这些化学信使的神经元的萌芽是对雌激素的反应，雌激素受体与这些生化信使的基因表达之间的关系以及伴随形成或伸长的过程已经被讨论过，特别是在男性和女性差异的背景下。通过免疫细胞化学结合雌激素受体抗体的使用，发现雌激素受体定位于细胞核中，这表明雌激素受体可以阻断细胞核中的免疫阳性反应。蛋白质在细胞质中合成，然后通过核孔与雄性和雌性大鼠下丘脑的微管转运至细胞核，并研究了神经突延伸的差异。雌激素诱导的编码细胞骨架特定蛋白（包括微管蛋白样分子）的变化已在下丘脑视前区得到证实。