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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

嗜铬细胞胞核内有糖皮质激素受体免疫反应。在培养液中加入地塞米松后，MAP-2的突起延长和免疫反应强度明显减弱。这些结果表明，嗜铬细胞向神经元的分化至少部分是由糖皮质激素通过其受体产生的体液因子决定的。糖皮质激素受体Cleary反义寡核苷酸的加入引起嗜铬细胞突起的显著延长，表明类固醇受体的蛋白质合成被对其…的反义抑制作用所阻断。免疫细胞化学研究表明，在下丘脑性二形区，雌激素诱导含有神经降压素、降钙素基因相关肽、甲硫氨酸脑啡肽、8胆囊素和多巴胺的免疫反应神经纤维的数量发生显著变化。更多含有这些化学信使的神经元是对雌激素的反应。讨论了雌激素受体和这些生化信使的基因表达与突起形成或延长的关系，特别是在性别差异的情况下。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。秋水仙碱处理阻断了细胞核内的免疫阳性反应，提示雌激素受体蛋白是在细胞质中合成的，随后通过核孔与微管一起运输到细胞核。取雄性和雌性大鼠下丘脑进行单层培养，观察其轴突伸展的差异。在这一点上，应该注意的是，发育和雌激素诱导的mRNAs编码细胞骨架特定蛋白的变化，包括微管蛋白样分子，已经在下丘脑视前区发现。较少