中国科考队员赴南极前后外周血淋巴细胞生物钟基因Clock和Bmall昼夜节律性转录解析

发布时间：2018-05-14 18:32

本文选题：Clock基因 + Bmal1基因　；参考：《苏州大学》2011年硕士论文

【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。产生昼夜节律的结构基础是分子计时器—昼夜节律生物钟(circadian clock)。对于主导节律调节的一些重要基因,称之为生物钟基因。机体的各种内在昼夜节律性变化可在外界环境因素的作用下得到重设性调节,以使机体活动与环境变化达成和谐统一。引发和调控昼夜节律生物钟的分子机制是在钟输入信号(如光照、温度等)的作用下,若干钟基因、钟相关基因和钟控基因及其蛋白产物通过转录—翻译—翻译后事件的相互衔接,组成钟振荡器的自身调控反馈环路,实现钟信号的精确输出。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24h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。上述基因产物以杂二聚体形式形成正向转录因子,与下游钟基因Per(Period)、Cry(Cryptochromes)或tim (Timeless)启动子区的E-box元件结合,激活一系列基因的转录和翻译,因此,Clock和Bmal1在生物钟的分子振荡机制中被认为起着极其重要的作用。然而,有关人外周血淋巴细胞核心钟基因Clock和Bmal1的昼夜节律性转录在南极大陆特殊环境下有着怎样的表现,至今未明,这正是本文所要探讨的内容。目的探讨赴南极前后科考队员外周血淋巴细胞核心钟基因Clock和Bmal1的昼夜节律性表达规律,旨在解析、拓展特殊环境下人类外周免疫钟运行的分子调控机制。方法 8名健康、男性中国南极科考队员志愿者,年龄24～30岁,平均25岁。赴南极前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,起床时间:7:00,睡眠时间(无光照期):23:00～7:00,早餐时间:7:30～8:00,午餐时间:11:30～12:00,晚餐时间:5:30～6:00;睡眠时光照强度0.1 Lux;受试者自由饮水,无烟酒嗜好,在过去6个月内未做过跨时区旅行和未接受过药物治疗,日常活动和饮食成分基本一致。随后在一昼夜内,随机选择2位受试者,每隔4 h抽取各自外周血6 ml,血样采集在4个不连续的随机昼夜内完成。分离其淋巴细胞,提取总RNA,逆转录为cDNA,采用实时荧光定量PCR方法,测定不同昼夜时点(zeitgeber time,ZT,共6个,每个时点n=8)每个样品中Clock和Bmal1基因的mRNA表达量,并以熔解曲线和凝胶电泳条带加以验证,通过余弦法和Clock Lab软件获取节律参数,并经振幅F检验分析是否存在昼夜节律性表达。选取相同8名男性科考队员,赴南极后生活一年,经历了极昼与极夜光照差异、季节与气候差异、气温与气压差异等自然环境变化。其昼夜模式生活条件、采血方式、取样时间、实验检测方法及数据统计学处理同上。结果 1.科考队员赴南极前外周血淋巴细胞核心钟基因Clock和Bmal1的昼夜表达 1.1在LD(16:8)光制下,科考队员外周血淋巴细胞钟基因Clock和Bmal1的mRNA表达呈现明显的昼夜节律性振荡(振幅F检验,P0.05); 1.2 Clock基因的峰值相位-335.85±13.80,表达振幅3.46±1.27,中值-11.40±1.59,峰时和谷时分别位于ZT22和ZT10,峰时与谷时mRNA水平分别为-7.95±2.55和-14.85±1.32; 1.3 Bmal1基因的峰值相位-307.12±108.17,表达振幅2.77±1.11 ,中值-5.50±1.32,峰时和谷时分别位于ZT20和ZT8,峰时与谷时mRNA水平分别为-2.74±0.71和-8.25±2.33; 1.4 Clock和Bmal1昼夜节律性转录的比较:两个基因LD(16:8)光制下在所检测的6个昼夜时点中表达水平均有明显差异(P0.05),Clock基因的表达水平较Bmal1基因降低;从昼夜节律性参数比较看出,两个基因表达的峰值相位、振幅无差异(P0.05),而Clock基因转录的中值水平以及峰时mRNA水平和谷时mRNA水平均降低(P0.05),Clock峰时和谷时比Bmal1推后约2小时(P0.05)。 2.科考队员赴南极后外周血淋巴细胞核心钟基因Clock和Bmal1的昼夜表达 2.1八名科考队员中分别有2人钟基因Clock、3人Bmal1的转录表达具有明显的昼夜节律性(振幅F检验,P0.05); 2.2二人Clock基因的峰值相位-42.28±5.27,表达振幅0.79±0.29,中值-9.02±0.24,峰时和谷时分别位于ZT3和ZT15,峰时与谷时mRNA水平分别为-8.23±0.53和-9.81±0.05; 2.3三人Bmal1基因的峰值相位-184.58±29.58,表达振幅1.04±0.65,中值-8.50±0.45,峰时和谷时分别位于ZT12和ZT24,峰时与谷时mRNA水平分别为-7.46±0.41和-9.54±1.04; 2.4 Clock和Bmal1昼夜节律性转录的比较:存在昼夜节律性表达的两个基因[Clock (n=2)和Bmal1 (n=3)]在所检测的6个昼夜时点(除了ZT14)的表达水平及其昼夜节律性参数变化均无显著性差异(P0.05); 2.5其他队员的上述两个基因不表现明显的昼夜节律性转录特征(振幅F检验,P0.05)。这些无节律表达队员的Clock基因,除昼夜时点ZT22以外,在其它各时点的表达量均低于Bmal1基因(P0.05)。 3.科考队员赴南极前、后外周血淋巴细胞核心钟基因Clock和Bmal1昼夜节律性转录的比较 3.1赴南极前、后钟基因Clock昼夜节律性表达的比较:赴南极后的Clock昼夜节律性表达,与赴南极前相比,除昼夜时点ZT18和ZT22以外,在其它时点的表达量均上调(P0.05)。队员赴南极后Clock基因的节律性表达振幅减小、中值增大、谷时mRNA水平增大(P0.05),峰时和谷时分别推迟约5个小时。 3.2赴南极前、后钟基因Bmal1昼夜节律性表达的比较:赴南极后的Bmal1昼夜节律性表达,与赴南极前相比,除昼夜时点ZT10和ZT14以外,在其它时点的表达量均下调(P0.05)。队员赴南极后Bmal1基因的节律性表达峰值相位推后,振幅、中值和峰时mRNA水平均下降(P0.05),峰时和谷时分别推迟约16个小时。结论 1.赴南极前,在LD(16:8)自然光制下,科考队员外周血淋巴细胞核心生物钟基因Clock和Bmal1的转录具有明显的昼夜节律性振荡特征;Clock转录的峰时和谷时分别位于ZT22和ZT10,Bmal1的峰时和谷时分别位于ZT20和ZT8;Clock基因各昼夜时点的表达水平、中值以及峰时mRNA水平和谷时mRNA水平较Bmal1基因的降低,Clock峰时和谷时比Bmal1推后约2小时。 2.赴南极后,经历各种自然环境变化,相同8名队员中多数人上述两个基因不再表现明显的昼夜节律性转录特征,Clock基因除昼夜时点ZT22以外,在其它各时点的表达量均低于Bmal1基因。 3.赴南极后,2人Clock基因、3人Bmal1基因的转录继续维持明显的昼夜节律性表达,其中Clock转录的峰时和谷时分别位于ZT3和ZT15,Bmal1的峰时和谷时分别位于ZT12和ZT24;两个基因在所检测的6个昼夜时点(ZT14除外)的表达水平及其昼夜节律性参数的相应变化基本一致。 4.与赴南极前相比,赴南极后的Clock昼夜节律性表达,除昼夜时点ZT18和ZT22以外,在其它时点均为上调,节律振幅减小、中值增大、谷时mRNA水平增大,节律峰时和谷时分别推迟约5个小时。 5.与赴南极前相比,赴南极后的Bmal1昼夜节律性表达,除昼夜时点ZT10和ZT14以外,在其它时点均为下调,节律峰值相位推后,振幅、中值、峰时mRNA水平均下降,节律峰时和谷时分别推迟约16个小时。
[Abstract]:The physiological function, biochemical metabolism and behavioral performance of human and animal often change regularly with the 24 h cycle. The rhythm of this change is called the circadian rhythm. The structure basis of the circadian rhythm is the molecular clock circadian clock (circadian clock). Some important genes governing the regulation of the dominant rhythm are called biological Zhong Ji. Because of the internal circadian rhythmic changes in the body, the body can be reset under the effect of external environmental factors so as to achieve a harmonious unity of the body's activities and environmental changes.
The molecular mechanism that triggers and regulates the circadian clock is a self-regulated feedback loop of clock oscillators, with the interaction of clock genes, clock related genes, bell controlled genes and their protein products through transcriptional translation events, under the action of the clock input signal (such as illumination, temperature, etc.). The core clock gene Clock and Bmal1, which is a positive regulating component in the loop, has been proved not only to express 24h rhythmically in the central clock, such as the suprachiasmatic nucleus and pineal body, but also in various peripheral clock tissues, including the blood cells. The above gene products form a positive transcription factor in the form of heterozygosity in the form of a heterozygous two polymer, and downstream. The binding of the E-box elements in the clock gene Per (Period), Cry (Cryptochromes) or Tim (Timeless) promoter activates the transcription and translation of a series of genes. Therefore, Clock and Bmal1 are considered to play an extremely important role in the molecular oscillation mechanism of the biological clock. However, there are the core clock genes of peripheral blood lymphocytes, Clock and Bmal1 circadian nodes. What is the expression of the law transcription in the special environment of the Antarctic continent?
objective
The circadian rhythmic expression of the core clock gene Clock and Bmal1 in the peripheral blood lymphocytes of the team members before and after Antarctica was explored to explore the molecular regulation mechanism of the operation of the peripheral immune clock in the special environment.
Method
8 healthy, male volunteers, aged 24~30 years old, average 25 years old. Before going to the south pole, the day and night rhythm model conditions (natural light system, 16 h-light: 8 h-dark cycle, LD) lived for 1 weeks: room temperature 25 + 1 oC, 7:00, sleep time (no light period): 23:00 to 1, lunch time: 1 to 1 At 2:00, dinner time: 5:30 to 0.1 Lux; the subjects were free to drink and smoke-free alcohol. In the last 6 months, no cross time travel and no drug treatment had been done. The daily activities and diet composition were basically the same. Then, 2 subjects were randomly selected for a day and night, and the blood samples were selected at 6 ml of their peripheral blood every 4 h. The collection was completed in 4 discontinuous random days. Separate its lymphocytes, extract the total RNA, reverse transcriptase cDNA, and use real-time fluorescent quantitative PCR method to determine the Clock and Bmal1 mRNA expressions of Clock and Bmal1 in every sample of day and night (zeitgeber time, ZT, altogether 6, each time point n=8), and tested by the melting curve and the gel electrophoresis strip. The circadian rhythm was obtained by cosine and Clock Lab software, and amplitude F test was used to analyze whether there was circadian rhythm expression.
8 men of the same men were selected to live for one year after they went to the South Pole. They experienced the difference between the day and night light, the difference between the season and the climate, the difference of air temperature and air pressure. The living conditions of the day and night, the way of collecting blood, the time of sampling, the method of testing and the data of statistical processing were the same.
Result
1. circadian expression of core clock genes Clock and Bmal1 in Antarctic peripheral blood lymphocytes of the expedition team members
1.1 under the light of LD (16:8), the mRNA expression of peripheral blood lymphocyte genes Clock and Bmal1 showed obvious diurnal rhythmic oscillations (amplitude F test, P0.05).
The peak phase of the 1.2 Clock gene is -335.85 + 13.80, the expression amplitude is 3.46 + 1.27, the median value is -11.40 + 1.59, the peak time and the valley are at ZT22 and ZT10 respectively. The mRNA level at peak time and valley time is -7.95 + 2.55 and -14.85 + 1.32, respectively.
The peak phase of the 1.3 Bmal1 gene is -307.12 + 108.17, the expression amplitude is 2.77 + 1.11, the median value is -5.50 + 1.32, the peak time and the valley are at ZT20 and ZT8 respectively. The mRNA level at peak time and valley time is -2.74 + 0.71 and -8.25 + 2.33, respectively.
1.4 Clock and Bmal1 circadian transcriptional comparison: two genes, LD (16:8), were significantly different in the 6 day and night time points detected (P0.05), and the expression level of the Clock gene was lower than that of the Bmal1 gene. From the circadian rhythmic parameters, the peak phase of the two gene expression, the amplitude was no difference (P0.05), and Clock (P0.05). The median level of gene transcription, peak mRNA and mRNA were decreased (P0.05), and Clock peak time and valley time were about 2 hours later than Bmal1 (P0.05).
2. the circadian expression of core clock genes Clock and Bmal1 of peripheral blood lymphocytes after Antarctic expedition to Antarctica
2.1 among the eight team members, there were 2 clock genes Clock and 3 Bmal1, respectively. The transcriptional expression of the 3 genes was obviously circadian (amplitude F test, P0.05).
The peak phase of the Clock gene of 2.2 two people is -42.28 + 5.27, the amplitude of the expression is 0.79 + 0.29, the median value is -9.02 + 0.24, the peak time and the valley are at ZT3 and ZT15 respectively, and the mRNA levels in peak time and valley are -8.23 + 0.53 and -9.81 + 0.05 respectively.
The peak phase of the Bmal1 gene of 2.3 three people is -184.58 + 29.58, the amplitude of the expression is 1.04 + 0.65, the median value is -8.50 + 0.45, the peak time and the valley are at ZT12 and ZT24 respectively, and the mRNA levels in peak time and valley are -7.46 + 0.41 and -9.54 + 1.04 respectively.
2.4 Clock and Bmal1 circadian transcriptional comparison: there were two genes [Clock (n=2) and Bmal1 (n=3), which had circadian rhythmic expression, and there was no significant difference in the level of expression and circadian rhythm of the 6 day and night points detected (P0.05).
2.5 of the two genes of other players did not exhibit significant diurnal rhythmic transcriptional characteristics (amplitude F test, P0.05). The Clock gene of these non rhythmic expression players, except for the day and night point ZT22, was lower than the Bmal1 gene at other time points (P0.05).
3. circadian rhythms of core clock genes Clock and Bmal1 in peripheral blood lymphocytes before and after Antarctic expedition to Antarctica
3.1 the day and night rhythmic expression of the post clock gene Clock before the South Pole: the circadian expression of Clock after the south pole, compared with the Antarctic, was up to up (P0.05) at other time points except for the day and night time ZT18 and ZT22 (P0.05). The amplitude of the rhythmic expression of the Clock gene decreased, the median value increased, and the mRNA level increased in the valley. Large (P0.05), peak hours and valley time were delayed by about 5 hours.
3.2 comparison of circadian rhythmic expression of the post clock gene Bmal1 before and after the Antarctic: the circadian expression of Bmal1 after the south pole, compared with the Antarctic, the expression of the expression at other time points, except for the day and night time ZT10 and ZT14, decreased (P0.05). The peak phase of the rhythmic expression of the Bmal1 gene after the team went to the south pole, the amplitude, the median and the peak mRNA The level decreased (P0.05), and the peak time and valley time were delayed by about 16 hours.
conclusion
1. before the south pole, under the LD (16:8) natural light system, the transcription of the transcription of the core biological clock gene Clock and Bmal1 of the peripheral blood lymphocytes of the members of the examination group had obvious circadian rhythmic oscillations; the peak time and the valley time of the Clock transcript were located at ZT22 and ZT10 respectively, and at the peak and the valley time of Bmal1, respectively, at the day and night time of the Clock gene. Flat, median and peak mRNA levels and valley time mRNA levels were lower than those of Bmal1 genes, and Clock peak time and valley time were about 2 hours later than Bmal1.
After 2. to the Antarctic, through various natural environment changes, the above two genes of the 8 members of the same team no longer showed significant circadian rhythmic transcriptional characteristics. The expression of Clock gene was lower than the Bmal1 gene at other time points except the day and night point ZT22.
3. after 3. to the Antarctic, the transcription of 2 Clock genes and 3 Bmal1 genes continued to maintain an obvious circadian expression, of which the peak time and the valley time of the Clock transcript were located in ZT3 and ZT15 respectively, and at the peak and the valley time of Bmal1 respectively in ZT12 and ZT24; the two genes were expressed in 6 day and night points (except ZT14) and the circadian rhythmic reference The corresponding changes in the number are basically the same.
4. compared with the Antarctic, the circadian rhythmic expression of Clock after the south pole, except for the day and night time ZT18 and ZT22, increased at other time points, decreased the amplitude of the rhythm, increased the median value, increased the mRNA level in the valley, and postponed about 5 hours when the rhythm peak and the valley time were delayed.
5. compared with the Antarctic, the circadian rhythmic expression of Bmal1 after the south pole, except for the day and night time ZT10 and ZT14, is down at the other time points, and the peak phase of the rhythm is pushed back, the amplitude, median and peak mRNA level all decrease, and the peak and valley time of the rhythm are delayed about 16 hours respectively.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R33

【参考文献】