| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
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.
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