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Aging of human circadian time keeping system and its management

人体生物钟计时系统的老化及其管理

基本信息

批准号：
13670980
负责人：
SUGAWARA Junya
金额：
$ 2.24万
依托单位：
Akita University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670980/
关键词：
clock genes circadian rhythem hPer1 hBmal1 Real Time-PCR hper1 RT-PCR

项目摘要

The aim of this study is to establish the gene expression analysis of several clock genes in human peripheral white blood cells to investigate the mechanism of hormonal transmission of circadian signal from biological clock to peripheral organs. We worked on two representative clock genes, human period1 homolog (hPer1: GenBank Accession number AB002107) and human BMAL1a homolog (hBmal1; #D89722), which are expected to possess the mutually opposite phase of gene expression profiles. We obtained whole white cells, or fractions of mononuclear and polynuclear peripheral cells by refined method from the 5 mL of whole peripheral blood collected from the healthy human volunteers. mRNA in each cell fraction was directly extracted using magnetic particle separator,and was converted into the first strand cDNA using AMV reverse transcriptase. We designed PCR primer and hybriprobe sets for each of hPer1, hBmall and β-actin (internal standard housekeeping gene) for the followlng Real Time-PCR. The hybriprobes, oligonucleotides, were designed to possess specific sequences to each target gene, and labelled with fluorescent dyes (fluorescein and LCRed640). After optimization of the PCR conditions, we could obtain stable and efficient amplification curve of these PCR products with high reproducibility. We could detect hPer1 as well as hBmal1 gene expressopn in both of the mononuclear and polynuclear peripheral cell fractions in all of 6 healthy volunteers whose sleep-wake cycles were controlled regularly. In mononuclear cells, both of rhe hPer1 and hBmal1 showed equivalent levels of gene expression. By contrast, hBmal1 showed 4 to 5-fold higher gene expression compared to that of the hPer1 in polynuclear cells. We could also obtain preliminary data that showed obvious diurnal variation in hPer1 gene expression in whole white cells in 4 healthy volunteers, which were analogous to that reported in the rodent suprachiasmatic nucleus.

本研究旨在建立人外周血白色细胞中几种生物钟基因的基因表达分析，探讨生物钟向外周器官传递昼夜节律信号的激素传递机制。我们研究了两个代表性的时钟基因，人类period 1同源物（hPer 1：GenBank登录号AB 002107）和人类BMAL 1a同源物（hBmal 1; #D89722），预计它们具有基因表达谱的相反相位。我们从健康志愿者采集的5 mL外周血中通过精制方法获得了全白色细胞或单核和多核外周血细胞的部分。使用磁性颗粒分离器直接提取每个细胞级分中的mRNA，并使用AMV逆转录酶将其转化为第一链cDNA。我们为hPer 1、hBmall和β-actin（内标管家基因）设计了PCR引物和杂交探针组，用于随后的真实的Time-PCR。杂交探针，寡核苷酸，被设计为具有每个靶基因的特异性序列，并用荧光染料（荧光素和LCREd 640）标记。通过对PCR反应条件的优化，获得了稳定、高效、重复性好的PCR产物扩增曲线。在6名睡眠-觉醒周期正常的健康志愿者的外周血单个核和多核细胞中均检测到hPer 1和hBmal 1基因的表达。在单核细胞中，rhe hPer 1和hBmal 1的基因表达水平相当。相比之下，hBmal 1在多核细胞中的基因表达比hPer 1高4 - 5倍。我们还可以获得的初步数据显示，在整个白色细胞在4名健康志愿者，这是类似于啮齿动物的视交叉上核的报道，hPer 1基因的表达明显的昼夜变化。