| Circadian rhythms can be found at levels of physiological function, biochemical metabolism and behavior change of living organism. Disorder of the rhythms may result in various diseases, such as jet lag caused by flight over different geographic time zones, dyssomnia induced by day and night shift-work, metabolic alteration and aging originated from hormonal parasecretion, mental ailment triggered by special rhythm impairment and so on. It is an important theoretical and practical sense to reveal the regulatory mechanism of circadian rhythm in terms of better understanding physiological, biochemical and behavioral activities of organism, and to explain pathologic process resulted from disturbance in circadian rhythm.Structures of biological clock include nucleus clock organization, i.e., suprachiasmatic nucleus (SCN), pineal gland (PG) and related peripheral tissues manipulating circadian rhythms. The molecular regulatory mechanisms of clock oscillation consist of clock signal input, several clock genes, clock-associated genes, clock-controlled genes and their protein, through the interconnection of intracellular transcription– translation - posttranslation event, to organize a fundamental molecular framework named autoregulatory feedback loop of the clock oscillator for accurate clock signal output. As the frndamental clock genes, Clock and Bmal1 are mainly expressed in the SCN, PG and retina. They could encode transcription factors which contain the structural domain, base helix-loop-helix (bHLH) and PER-ASNT-SIM (PAS). Common ligand product of two genes, CLOCK:BMAL1 intranuclearly shape a heterodimer protein as a positive regulator, combining with the E-box element in the promoter region of the clock genes such as Period (Per), cryptochromes (Cry) or timeless (Tim), and then activate these genes transcription and translation. Now, circadian expressive patterns of the clock genes, Clock and Bmal1 in different central tissue in the mammals have been reported. Study on circadian rhythmic expressions and photoresponses of two genes in the peripheral clock tissue, especially, in human peripheral blood lymphocytes, however, is still open so far, which is an aim of present study.Objective: This study was conducted to investigate the circadian rhythmic expressions of clock genes, Clock and Bmal1 in the human peripheral blood lymphocytes and to better understand molecular regulatory mechanism of the peripheral immuneclock action.Methods: 10 healthy male volunteers aged from 24～30 years (mean age 25 years) previously lived under the circadian model condition (natural light regime, 16 h-light : 8 h-dark cycle, LD) for one week. room temperature 25±1 oC, time out of bed 7:00, bedtime (no photoperiod) 23:00～7:00, breakfast time 7:30～8:00, lunch time 11:30～12:00 and supper time 5:00～6:00. Intensity of illumination was less than 0.1 Lux during sleeping time and subjects drank water freely, and had no smoking and alcohol drinking but the same food intake and daily activities. Then every subject was sampled for the peripheral blood 6 ml every 4 hours in a circadian day. Lymphocytes were separated from blood and the total RNA was extracted from each sample. The quantitative real time reverse transcription polymerase chain reaction (RT-PCR) was used to determine the temporal changes in mRNA levels of Clock gene and Bmal1 gene during different zeitgeber time (ZT, n=6 for 24 hours, each time point was n=10). Gel electrophoresis and dissociation curve analysis were used for data double check. The data circadian parameters were obtained and analyzed by both the cosine function, Clock Lab software and the amplitude F test to reveal the genes` circadian rhythm in the LD (16:08) condition.Results:1. Under the LD (16:08) light regime, both the Clock and Bmal1 mRNA transcription in healthy adult human peripheral blood lymphocytes displayed a robust circadian oscillation (amplitude F test, P < 0.05). 2. For the Clock gene, circadian parameters peak phase was -161.70±17.36, amplitude was 3.08±1.38, mesor was 11.70±1.58, peak time was ZT13, trough time was ZT1, mRNA level at peak was 14.77±1.26 and mRNA level at trough was 8.63±2.67.3. For the Bmal1 gene, circadian parameters peak phase was -177.55±23.48, amplitude was 2.67±1.23, mesor was 5.50±1.52, peak time was ZT12, trough time was ZT24, mRNA level at peak was 8.16±2.67 and mRNA level at trough was 2.83±0.71.4. Under the LD (16:08) condition, in comparison with Clock gene, the expressive levels at different time points, mesor, mRNA level at peak and at trough of the Bmal1 gene in the adult human peripheral blood lymphocytes were correspondingly smaller (P<0.05) but parameters peak phase, amplitude, peak time and trough time of Bmal1 gene expression were similar to those of the Clock gene (P>0.05).Conclusion:1. Under the LD (16:08) light regime, clock genes, the Clock and Bmal1 mRNA transcriptions in healthy adult human peripheral blood lymphocytes possess a remarkable circadian oscillation with a peak at ZT13 for Clock gene or ZT12 for Bmal1 gene and a trough at ZT1 for Clock gene or ZT24 for Bmal1 gene.2. Under the LD (16:08) condition, the peak phase, amplitude, peak time and trough time of the two genes circadian expressions in the adult human peripheral blood lymphocytes are similar to each other, Which evidence that the Clock and Bmal1 show a synchronous circadian rhythmic transcription characteristics in human peripheral blood lymphocytes.3. Under the LD (16:08) light regime, the expressive levels at different time points, mesor, mRNA level at peak and at trough of the Bmal1 gene in the adult human peripheral blood lymphocytes are correspondingly smaller than those of Clock gene.
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