小鼠外周血白细胞节律相关基因 cry2，per2，timeless,rev-erb表达的近日节律性研究

摘要

目的：探讨在12 h光照/12 h黑暗（12L/12D）光暗循环条件下，小鼠外周血白细胞中相关基因cry2，per2，time-less，rev-erb近日节律性表达特点。方法用完全随机分组法将C57/BL6小鼠分成6组，每组对应一个时相点，在12L/12D条件下词养4周后分别在6个时相点（9：00，13：00，17：00，21：00，1：00，5：00）取外周血并分离白细胞。抽提总RNA，采用实时荧光定量PCR技术检测小鼠外周血白细胞cry2，per2，timeless，rev-erb 4种节律基因的mRNA水平，用余弦函数拟合节律参数，并经振幅f检验分析是否存在近日节律。结果 cry2，per2，timeless，rev-erb 4种基因mRNA存在明显的近日节律性，各基因的峰值明显高于谷值（P＜0．01）；per2峰值相位时间在4：00左右，而cry2，timeless，rev-erb基因的峰值时间在11：00-12：00；从峰值时间和震荡幅度看，cry2，timeless，rev-erb峰值时间比per2超前，振幅比per2高，cry2、timeless、rev-erb的峰值时间、振幅大小比较接近。结论 C57/BL6小鼠外周血白细胞4种基因cry2，per2，timeless，rev-erb的mRNA存在明显的近日节律性。 cry2，timeless， rev-erb在负反馈中的作用强于per2，而cry2，timeless，rev-erb在负反馈中的作用相似。该研究为深入理解哺乳动物的免疫功能昼夜波动打下了基础，也为诊断、治疗免疫功能紊乱相关疾病提供了靶目标。但这4种基因如何调控外周血中的免疫细胞，尚需进一步深入研究。%Objective To investigate features of circadian expressions of related genes of cry 2, per2, timeless and rev-erb in peripheral white blood cells in mice under the 12 h-light and 12 h-dark cycle conditions .Methods Forty-eight male C57/BL6 mice were randomly divided into 6 groups, with each group directed to a corresponding time point .The animals were kept under the light reg-imen of LD cycle (12L/12D) for four weeks.Then, peripheral blood samples were taken at 6 time points (9:00, 13:00, 17:00, 21:00,1:00, 5:00) and white blood cells were separated .Total RNA was extracted from the white blood cells and mRNA levels of each gene (cry2, per2, timeless and rev-erb) were detected by reverse transcription polymerase chain reaction (real time PCR) at dif-ferent time points .The data were analyzed both by the cosine function software and the amplitude F test to investigate the circadian rhythm.Results Under the light regimen of LD cycle , the four genes in white blood cells showed a significant circadian oscillation , with the apexes of different genes being obviously higher than the troughs (P<0.01).The apex of per2 was at about 4:00, while the a-pex of the other three genes were at about 11:00-12:00.From the standpoints of apex time and oscillation , the apex time of cry2, time-less and rev-erb was before that of per2, with a higher level of amplitude than that of per 2.The apex time of cry2, timeless, rev-erb mRNA was before that of per2, with the amplitude being higher than that of per 2.However,the mRNAs of cry2, timeless and rev-erb showed a similar apex time and amplitude .Conclusion Research findings demonstrated that the expressions of the 4 genes in peripher-al white blood cells showed an obvious circadian rhythm .It seemed that the effects of cry 2, timeless and rev-erb were stronger than that of per2 in the negative regulation , while the effect of rev-erb was similar in the negative regulation .This research might not only be helpful to the deep understanding of the circadian fluctuation of immune function in mammals , but provide a target for diagnosis and therapy of immune disorders as well .Further detail study was required to fully understand how these 4 genes regulate immune cells in the peripheral blood .