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Molecular basis of the circadian rhythm for peptides mRNA

肽 mRNA 昼夜节律的分子基础

基本信息

批准号：
02807016
负责人：
ISOBE Yoshiaki
金额：
$ 1.09万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02807016/
关键词：
Circadian rhythm Vasoactive intestinal peptide Vasopressin Development Gastrin releasing peptide Culture Intracellular Ca Arginine Vasopressin Suprachiasmatic nucleus Vasoactive intestinal peptide Gastrin releasing peptide Circadian

项目摘要

In mammals, a pacemaker of circadian rhythms is considered to be located in the suprachiasmatic nuclei(SCN). We examined the role of peptides contained in the SCN of rats for molecular bases. Vasoactive intestinal peptide (VIP) and gastrin releasing peptide are colocalized in ventrolateral area of the SCN were measured by ELA method under the light dark cycle. No clear circadian rhythm of VIP and GRP was not observed in the 2-days-blind rats, though the VIP and GRP in intact rats showed higher concentration during the subjective night and day, respectively. The Northern analysis of mRNA was done to know the transcriptional stage in the synthesis of these peptides The SCN of 4-6 brains were microdissected every 4hr throughout a day and total RNA was extracted by the phenol-chloroform method. VIP mRNA levels showed higher during the dark than in the light period. This rhythmicity was not observed in the cortex. The results suggest possible involvement of neuropeptides in circadian genera … More tion at their messenger RNA levels.Beside, to know the ontogeny of these peptides oscillation for circadian clock, the development of these peptides were analyzed. The concentration of VIP during the dark time began to higher in the dark period than in light one from the post natal day 4 and succeeded till the wean.To establish an in vitro system to observe the SCN function for a long duration, we used a dissociated cell culture system to study the cellular mechanisms of SCN. Dispersed cells of SCN were obtained from P-1 day pups whose mother were kept under light-dark cycle. Arginine-vasopressin (AVP), plays important role for the output signal form the SCN in vivo, in the culture medium were measured. The release of AVP from these cells showed a accurate circadian oscillation. The circadian rhythms of AVP were reversed in phase when the cell were prepared 12hr out of phase from the pups kept under the light-dark cycle. Some neurons were certified by AVP antibody using the ABC method. The dispersed culture system provides a useful model for elucidating the mechanism of pacemaker. We, now, are developing the Organotipic slice culture having a number of advantage of structural organization. Less

在哺乳动物中，昼夜节律的起搏器被认为位于视交叉上核（SCN）。我们检查了大鼠 SCN 中所含肽的分子基础作用。血管活性肠肽(VIP)和胃泌素释放肽共定位于SCN腹外侧区，采用ELA方法在明暗循环下进行测量。尽管完整大鼠中的 VIP 和 GRP 在主观夜间和白天分别表现出较高的浓度，但在 2 天盲法大鼠中未观察到明显的 VIP 和 GRP 昼夜节律。进行 mRNA 的 Northern 分析，以了解这些肽合成的转录阶段。全天每 4 小时对 4-6 个大脑的 SCN 进行显微解剖，并通过苯酚-氯仿方法提取总 RNA。 VIP mRNA 水平在黑暗期间比在光照期间更高。在皮层中没有观察到这种节律性。结果表明，神经肽可能在其信使 RNA 水平上参与昼夜节律生成。此外，为了了解这些肽在生物钟振荡中的个体发育，我们对这些肽的发育进行了分析。暗期VIP浓度从产后第4天开始，暗期高于亮期，一直持续到断奶。为了建立长期观察SCN功能的体外系统，我们使用离解细胞培养系统来研究SCN的细胞机制。 SCN分散细胞从P-1日龄幼鼠中获得，其母亲置于明暗循环下。精氨酸加压素（AVP）对于体内SCN的输出信号起着重要作用，在培养基中进行了测量。这些细胞释放的 AVP 显示出精确的昼夜节律振荡。当细胞制备时，AVP 的昼夜节律与保持在明暗循环下的幼崽异相 12 小时。一些神经元通过 AVP 抗体使用 ABC 方法进行认证。分散培养系统为阐明起搏器的机制提供了有用的模型。我们现在正在开发具有许多结构组织优势的 Organotipic 切片培养物。较少的