Cell Biological Approach to the Elucidation of Intercellular Messenger Molecules

用原位杂交组织化学方法半定量地研究了肽、胺和类固醇的前体和合成酶的mRNA的定位和变化。此外，他们从mRNA翻译的蛋白质进行了分析，在组织切片的帮助下，修改的免疫细胞化学结合氚化抗体。在该项目的第一年，我们试图专注于技术问题的开发和建立，包括mRNA及其蛋白质定量参数的标准化。本研究所用的mRNA和蛋白质为加压素、催产素、CRF、脑啡肽、CGRP、NPY和MAP 2的前体，酪氨酸羟化酶、色氨酸羟化酶和胆碱乙酰转移酶的酶，以及5-羟色胺、雌激素、孕酮、睾酮和糖皮质激素的受体蛋白。用计算机辅助DNA合成仪设计并制备了这些材料的探针， ...更多信息 在第二年，我们进行了包括生理和药理操作的实验，作为盐负荷、脱水、卵巢切除、去势、类固醇治疗、秋水仙碱注射和利血平治疗。在此条件下，研究了下丘脑核神经元和肾上腺内分泌细胞中每种mRNA的变化。与此同时，与对照组相比，在操作的组织上检查免疫细胞化学变化。在我们看来，这些信使和蛋白质是基因表达、生产、运输和生物活性分子释放的细胞内机制的良好指示剂。此外，将后垂体、脊髓、中脑区域和皮肤植入大脑的第三脑室或眼前房，以阐明细胞靶识别的特异性。其中一项结果显示了几种神经元对原靶区的特异性神经再支配模式。少

The localizations and changes of mRNAs of the precursors and synthesizing enzymes for peptides, amines, and steroids were semiquantitatively investigated by using an in situ hybridization histochemistry. In addition, their translated proteins from mRNAs were analyzed on tissue sections with the aid of a modified immunocytochemistry in conjunction with tritiated antibodies. In the first year of this project, we tried to focus on the development and establishment of technical matters, including the standardization of parameters for the quantification of mRNAs and their proteins on the sections. The mRNAs and proteins used in this project were the precursors of vasopressin, oxytocin, CRF, enkephalin, CGRP, NPY and MAP2, and enzymes of tyrosine hydroxylase, tryptophan hydroxylase and choline acetyltransferase, and receptor proteins of serotonin, estrogen, progesterone, testosterone and glucocorticoids. Probes for these materials were designed and made by computer-assisted DNA synthesizer t … More o minimize the homologous sequence to their similar mRNAs.In the second year we carried out experiments which include physiological and pharmacological manipulations such as salt loading, dehydration, ovariectomy, castration, steroid treatment, colchicine injiction and reserpine treatment. Under such conditions, each mRNA in neurons of the hypothalamic nuclei and in endocrine cells of adrenal gland was investigated on its changes. At the same time immunocytochemical changes were examined on the manipulated tissue in comparison with the control. It seems to us that these messengers and proteins are good indicator for the intracellular mechanisms of the gene expression, production, transport, and release of bioactive molecules. Furthermore posterior pituitaries, spinal cords, midbrain regions and skins were implanted into the third ventricle of the brain or anterior eye chamber to elucidate the specificity of cellular target recognition. One of our results showed specific reinnervation pattern of several kinds of neurons to the original target areas. Less