【作者】 张文娟；

【导师】 王国卿；

【作者基本信息】 苏州大学 ， 生理学， 2010， 硕士

【摘要】 人和动物的生理机能、生化代谢、行为表现等常以24 h为周期发生规律性变动,这种变化的节律称为昼夜节律。从细菌到哺乳动物体内都有控制昼夜节律的装置,称为生物钟。生物钟作为定时系统,既使机体的生命活动和行为以24 h为周期发生规律性变动,又能在外界环境因素的作用下重设节律输出,以使机体活动与环境变化达成和谐统一。昼夜节律的分子生物学基础是一系列钟基因的转录-翻译活动形成的振荡反馈环路,其中作为环路中正向调节成分的核心钟基因Clock和Bmal1,已被证实不但24 h节律性表达于中枢钟组织如视交叉上核与松果体,而且也表达于包括血细胞在内的各种外周钟组织,上述基因产物以异二聚体形式形成正向转录因子,在生物钟的分子振荡机制中起着极其重要的作用。关于人类外周血淋巴细胞核心钟基因Clock和Bmal1的昼夜节律性表达外观,已有报道进行了mRNA水平的研究,而在翻译水平上人类外周血淋巴细胞CLOCK和BMAL1蛋白—这对正向转录因子的昼夜节律性表达规律及其光反应性有着怎样的表现,至今尚待明确,这正是本文所要探讨的内容。目的:探讨成年人外周血淋巴细胞CLOCK蛋白和BMAL1蛋白的昼夜节律性表达规律,旨在进一步从蛋白水平上解析、丰富人类外周免疫钟运行的分子调控机制。方法:8名健康男性志愿者,年龄18～22岁,平均20岁。预先在昼夜节律模式条件(自然光制,16 h-light : 8 h-dark cycle, LD )下生活1周,室温25±1 oC。受试者自由饮水,无烟酒嗜好,日常活动和饮食成分基本一致。随后在一昼夜内每隔4 h抽取各受试者外周血12 ml,分离淋巴细胞,提取总蛋白,用BCA(bicinchonininc acid)法测定蛋白浓度,采用Western Blot方法,检测不同昼夜时点(zeitgeber time,ZT,共6个,每个时点n=8)样品中CLOCK和BMAL1蛋白的表达量,通过余弦法和Clock Lab软件获取节律参数,并经振幅检验分析其是否存在昼夜节律。结果:1. LD(16:8)光制下,正常成年人外周血淋巴细胞核心转录因子钟蛋白CLOCK和BMAL1的表达呈现明显的昼夜节律性振荡变化(振幅F检验,P<0.05)。2. CLOCK蛋白的峰值相位-40.94±13.63,表达振幅0.50±0.23,中值2.91±0.23,峰值和谷值时间分别位于ZT3和ZT15,蛋白峰时水平3.40±0.34,谷时水平2.41±0.29。3. BMAL1蛋白的峰值相位-88.49±12.23,表达振幅0.31±0.08,中值2.85±0.49,峰值和谷值时间分别位于ZT6和ZT18,蛋白峰时水平3.15±0.54,谷时水平2.55±0.46。4. LD(16:8)光制下,人外周血淋巴细胞BMAL1蛋白在不同昼夜时点的表达水平、振幅、中值、蛋白峰时水平和谷时水平,分别与CLOCK蛋白相比,均无显著性差别(P>0.05);但其峰值相位、峰值时间和谷值时间,分别与CLOCK蛋白相比,均有显著性差异(P<0.05),相位延迟3小时。结论:1. LD(16:8)光制下,成年人外周血淋巴细胞核心钟蛋白CLOCK和BMAL1的表达呈现明显的昼夜节律性振荡,CLOCK蛋白的峰值和谷值时刻分别位于ZT3和ZT15,BMAL1蛋白的峰值和谷值时刻分别位于ZT6和ZT18。2. LD(16:8)光制下,人外周血淋巴细胞核心钟蛋白CLOCK和BMAL1在不同昼夜时点的表达水平、振幅、中值、蛋白峰时水平和谷时水平相一致。3. LD(16:8)光制下,人外周血淋巴细胞核心钟蛋白BMAL1昼夜节律性表达的峰值相位、峰值时间和谷值时间,相对钟蛋白CLOCK而言,其节律相位滞后3小时。更多还原

【Abstract】 Circadian rhythms can be found at levels of physiological function, biochemical metabolism and behavior change for 24 h which is the regular cycle in human and animals. Signal output of the rhythm in vivo, generated by endogenous biological clocks, not only regulate various activities spontaneously but also receive resetting by the entrainment with external environmental factors named zeitgeber, in order to make body activities be in accordance with environmental changes. 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. Of core clock genes, the Clock and Bmal1 genes whose product i.e. heterodimer has been together regarded as a crucial positive transcription regulator in the loop, have been evidenced the 24-hour rhythmic expressions not only in central clock tissues such as the suprachiasmatic nucleus (SCN) and the pineal gland (PG), but also in various peripheral clock tissues including blood cells.The research of core clock genes Clock and Bmal1 circadian expressive profile has been reported at mRNA levels in human peripheral blood lymphocytes, however, study on circadian rhythmic expressions and photoresponses of CLOCK and BMAL1 proteins in human peripheral blood lymphocytes is not clear so far, which is an aim of present study.Objective: This study was conducted to investigate the circadian rhythmic expressions of clock proteins, CLOCK and BMAL1 in the human peripheral blood lymphocytes and to better understand molecular regulatory mechanism of the peripheral immuneclock action.Methods: 8 healthy male volunteers aged from 18~22 years (mean age 20 years) previously lived under the circadian model condition (natural light regime, 16 h-light : 8 h-dark cycle, LD) for a week, room temperature 25±1 oC. All 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 12 ml every 4 hours in a circadian day. Lymphocytes were separated from blood and the total proteins were extracted from each sample. The method of bicinchonininc acid (BCA) was used to examine the total proteins concentration and the Western Blot was conducted to determine the temporal changes of CLOCK and BMAL1 protein levels during different zeitgeber time (ZT, n=6 for 24 hours, each time point was n=8). The data of circadian parameters were obtained and analyzed by both the cosine function, Clock Lab software and the amplitude F test to reveal two protein circadian rhythm in the LD (16:08) condition.Results:1. Under the LD (16:08) light regime, the core transcription factors CLOCK and BMAL1 in healthy adult human peripheral blood lymphocytes displayed a robust circadian oscillatory expressions (amplitude F test, P < 0.05).2. For the CLOCK protein expression within 24 hs, circadian parameters peak phase was -40.94±13.63, amplitude was 0.50±0.23, mesor was 2.91±0.23, peak time was ZT3, trough time was ZT15, protein level at peak was 3.40±0.34 and protein level at trough was 2.41±0.29.3. For the BMAL1 protein expression within 24 hs, circadian parameters peak phase was -88.49±12.23, amplitude was 0.31±0.08, mesor was 2.85±0.49, peak time was ZT6, trough time was ZT18, protein level at peak was 3.15±0.54 and protein level at trough was 2.55±0.46.4. Under the LD (16:08) light condition, the expression level at different circadian time points, amplitude, mesor, protein level at peak time and protein level at trough time of the BMAL1 protein in human peripheral blood lymphocytes, compared with that of the CLOCK protein respectively, had no significant difference obtained (P> 0.05), however, the peak phase, peak time and trough time of the BMAL1 protein expression within 24 hs showed 3 hours later than those of the CLOCK protein (P <0.05).Conclusion:1. Under the LD (16:08) light regime, expressions of the CLOCK and BMAL1 proteins in healthy adult human peripheral blood lymphocytes possess a remarkable circadian oscillation with a peak at ZT3 and a trough at ZT15 for the CLOCK protein , and with a peak at ZT6 and a trough at ZT18 for the BMAL1 protein.2. Under the LD (16:08) light condition, the expression level at different circadian time points, amplitude, mesor, protein level at peak time and protein level at trough time of the CLOCK protein in healthy adult human peripheral blood lymphocytes is corresponding to that of the BMAL1 protein, respectively.3. Under the LD (16:08) light regime, the peak phase, peak time, trough time of the BMAL1 protein circadian expression in healthy adult human peripheral blood lymphocytes, compared with those of the CLOCK protein, are delayed 3 hours.更多还原