成年人外周血淋巴细胞生物钟基因Clock和Bmall昼夜表达规律的研究
发布时间：2018-08-03 11:01
【摘要】： 机体的生理功能、生化代谢、行为改变等均表现昼夜节律现象。这种节律的紊乱可导致多种疾病,如飞行跨越不同地理时区的时差反应、昼夜轮班制作业引起的睡眠障碍、激素分泌异常诱发的代谢失调与提前衰老、特殊节律缺陷导致的神经精神疾病等。揭示昼夜节律的发生原因和调节机制,对解释正常机体的生理、生化和行为现象以及因昼夜节律紊乱而引发的病理生理过程,乃至提出相应的防治措施等都有重要的理论意义和应用价值。 调控昼夜节律的生物钟包括中枢钟组织视交叉上核(Suprachiasmatic nucleus, SCN)、松果体(Pineal gland, PG)和各种外周钟组织。其分子机制是在钟输入信号的作用下,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。作为重要的钟基因,Clock和Bmal1主要表达于SCN、松果体和视网膜,可编码具有碱性螺旋—环—螺旋(base helix-loop-helix, bHLH)以及PAS( PER-ASNT-SIM )结构域家族的转录因子,互为配体的共同产物—CLOCK:BMAL1蛋白在细胞核内形成杂二聚体,作为正向调节子与下游钟基因Per(Period)、Cry(cryptochromes)或tim (timeless)启动子区的E-box元件结合,激活这些基因的转录和翻译。 有关哺乳类中枢不同部位钟基因Clock和Bmal1昼夜表达规律的研究,已有报道。而外周钟组织尤其是人类外周淋巴细胞Clock和Bmal1的昼夜节律性表达及其光反应性还不清楚,这正是本研究所要探讨的内容。 目的: 探讨成年人外周血淋巴细胞钟基因Clock和Bmal1的昼夜节律性定量表达规律,旨在进一步解析外周免疫钟运行的分子调控机制。 方法: 10名健康男性志愿者,年龄24～30岁,平均25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,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:5:00～6:00;睡眠时光照强度0.1 Lux;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6 ml,分离淋巴细胞,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=10)样品中Clock和Bmal1基因的mRNA表达量,并以溶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,并经振幅检验分析是否存在昼夜节律。 结果: 1. LD(16:8)光制下,正常成年人外周血淋巴细胞钟基因Clock和Bmal1的mRNA转录呈现明显的昼夜节律性振荡(振幅F检验,P0.05); 2. Clock基因的峰值相位-161.70±17.36,表达振幅3.08±1.38,中值11.70±1.58,峰值和谷值时间分别位于ZT13和ZT1,峰值相与谷值相mRNA水平分别为14.77±1.26和8.63±2.67; 3. Bmal1基因的峰值相位-177.55±23.48,表达振幅2.67±1.23,中值5.50±1.52,峰值和谷值时间分别位于ZT12和ZT24,峰值相与谷值相mRNA水平分别为8.16±2.67和2.83±0.71; 4. LD(16:8)光制下,人外周血淋巴细胞Bmal1基因在不同昼夜时点的表达水平较Clock基因降低(P0.05),两个基因转录的峰值相位、振幅、峰值时间和谷值时间相一致(P0.05),而Bmal1基因转录的中值水平以及峰值相的mRNA水平和谷值相的mRNA水平降低(P0.05)。 结论: 1. LD(16:8)光制下,成年人外周血淋巴细胞生物钟基因Clock和Bmal1的转录振荡具有明显的昼夜节律性,Clock基因转录的峰值和谷值时间分别位于ZT13和ZT1,Bmal1基因的峰值和谷值时间分别位于ZT12和ZT24; 2. LD(16:8)光制下,人外周血淋巴细胞生物钟基因Clock和Bmal1昼夜节律性表达的峰值相位、振幅、峰值时间和谷值时间相一致,二者存在同步化的昼夜节律性转录特征; 3. LD(16:8)光制下,人外周血淋巴细胞Bmal1基因不同昼夜时点的表达水平、中值、以及峰值相的mRNA水平和谷值相的mRNA水平较Clock基因的降低。
[Abstract]:The physiological functions, biochemical metabolism and behavioral changes of the body all show circadian rhythms. The disorder can lead to a variety of diseases, such as the time difference of flight across different geographic time zones, sleep disorders caused by day and night shift production, abnormal secretion of metabolic disorders and premature senescence induced by hormone secretion, and the deity caused by special rhythm defects. It has important theoretical significance and value to explain the causes and regulation mechanism of the circadian rhythm, and to explain the physiological, biochemical and behavioral phenomena of the normal body, the pathophysiological process caused by the disorder of the circadian rhythm, and even the corresponding preventive measures.
The circadian rhythms include Suprachiasmatic nucleus (SCN), Pineal gland (PG) and various peripheral clocks in the central clock. The molecular mechanism of the clock genes, clock related genes, bell controlled genes and their protein products through transcriptional translation - translations under the action of the clock input signal As an important clock gene, Clock and Bmal1 are mainly expressed in SCN, pineal and retina, which can encode transcriptional causes of the family of alkaline spiral - loop - helix (base helix-loop-helix, bHLH) and PAS (PER-ASNT-SIM) domain. The CLOCK:BMAL1 protein, a common product of the ligand, forms a heteropolymer in the nucleus, which acts as a positive regulator in conjunction with the E-box elements in the promoter region of the downstream clock gene Per (Period), Cry (cryptochromes) or Tim (timeless) and activates the transcription and translation of these genes.
The study of the day and night expression of Clock and Bmal1 genes in different parts of the mammalian center has been reported. The circadian rhythmic expression and light reactivity of the peripheral clock tissue, especially the human peripheral lymphocyte Clock and Bmal1, and the light reactivity are not clear. This is the internal capacity to be discussed in this study.
Objective:
To explore the regularity of circadian rhythmic quantitative expression of Clock and Bmal1 in peripheral blood lymphocyte clocks in adult peripheral blood, and to further analyze the molecular regulation mechanism of the operation of the peripheral immune clock.
Method:
10 healthy male volunteers, aged 24~30 years, average 25 years of age, in advance under the circadian rhythm model conditions (natural light, 16 h-light: 8 h-dark cycle, LD) for 1 weeks: room temperature 25 + 1 oC, 7:00, sleep time (no light period): 23:00 ~ dinner time: dinner time: supper time: The intensity of sleep time was 0.1 Lux; the subjects were free drinking water, smokeless alcohol hobby, daily activity and diet composition basically identical. Then the peripheral blood of the subjects was 6 ml every 4 h every day and night, the lymphocyte was separated, the total RNA was extracted and cDNA was extracted. The time point of day and night (zeitgeber time, ZT, total 6) was measured by the real-time fluorescence quantitative PCR method. The mRNA expression of the Clock and Bmal1 genes in the samples at each time point n=10) was verified by the dissolution curve and the gel electrophoresis strip. The rhythmic parameters were obtained by the cosine method and the Clock Lab software, and the circadian rhythm was analyzed by the amplitude test.
Result:
1. Under LD (16:8) light, the transcription of Clock and Bmal1 mRNA in normal adult peripheral blood lymphocytes showed obvious circadian oscillation (amplitude F test, P 0.05).
The peak phase of the 2. Clock gene is -161.70 + 17.36, the amplitude of the expression is 3.08 + 1.38, the median value is 11.70 + 1.58, the peak and the valley time are at ZT13 and ZT1 respectively, and the peak phase and the valley value mRNA level are 14.77 + 1.26 and 8.63 + 2.67, respectively.
The peak phase of the 3. Bmal1 gene is -177.55 + 23.48, the amplitude of the expression is 2.67 + 1.23, the median value is 5.50 + 1.52, the peak and the valley time are at ZT12 and ZT24 respectively, and the peak phase and the valley value mRNA level are 8.16 + 2.67 and 2.83 + 0.71, respectively.
In 4. LD (16:8) light, the expression level of Bmal1 gene in human peripheral blood lymphocytes at different day and night time was lower than that of Clock gene (P0.05). The peak phase, amplitude, peak time and valley time of the two gene transcription were the same (P0.05), while the level of the median of Bmal1 gene transcription, the mRNA level of the peak phase and the mRNA level of the valley value were reduced. (P0.05).
Conclusion:
Under 1. LD (16:8) light, the transcriptional oscillation of adult peripheral blood lymphocyte clocks gene Clock and Bmal1 has obvious circadian rhythms. The peak and valley time of Clock gene transcription are located at ZT13 and ZT1 respectively. The peak and valley time of Bmal1 gene are at ZT12 and ZT24 respectively.
In 2. LD (16:8) light, the peak phase, amplitude, peak time and valley time of the circadian clock genes of human peripheral blood lymphocyte clocks gene Clock and Bmal1 were the same as the valley time, and the two circadian rhythmic transcriptional characteristics were present.
Under 3. LD (16:8) light, the expression level of Bmal1 gene in human peripheral blood lymphocytes at different day and night time, the median value, the mRNA level of the peak phase and the mRNA level of the valley value phase were lower than those of the Clock gene.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R33
本文编号：2161538
[Abstract]:The physiological functions, biochemical metabolism and behavioral changes of the body all show circadian rhythms. The disorder can lead to a variety of diseases, such as the time difference of flight across different geographic time zones, sleep disorders caused by day and night shift production, abnormal secretion of metabolic disorders and premature senescence induced by hormone secretion, and the deity caused by special rhythm defects. It has important theoretical significance and value to explain the causes and regulation mechanism of the circadian rhythm, and to explain the physiological, biochemical and behavioral phenomena of the normal body, the pathophysiological process caused by the disorder of the circadian rhythm, and even the corresponding preventive measures.
The circadian rhythms include Suprachiasmatic nucleus (SCN), Pineal gland (PG) and various peripheral clocks in the central clock. The molecular mechanism of the clock genes, clock related genes, bell controlled genes and their protein products through transcriptional translation - translations under the action of the clock input signal As an important clock gene, Clock and Bmal1 are mainly expressed in SCN, pineal and retina, which can encode transcriptional causes of the family of alkaline spiral - loop - helix (base helix-loop-helix, bHLH) and PAS (PER-ASNT-SIM) domain. The CLOCK:BMAL1 protein, a common product of the ligand, forms a heteropolymer in the nucleus, which acts as a positive regulator in conjunction with the E-box elements in the promoter region of the downstream clock gene Per (Period), Cry (cryptochromes) or Tim (timeless) and activates the transcription and translation of these genes.
The study of the day and night expression of Clock and Bmal1 genes in different parts of the mammalian center has been reported. The circadian rhythmic expression and light reactivity of the peripheral clock tissue, especially the human peripheral lymphocyte Clock and Bmal1, and the light reactivity are not clear. This is the internal capacity to be discussed in this study.
Objective:
To explore the regularity of circadian rhythmic quantitative expression of Clock and Bmal1 in peripheral blood lymphocyte clocks in adult peripheral blood, and to further analyze the molecular regulation mechanism of the operation of the peripheral immune clock.
Method:
10 healthy male volunteers, aged 24~30 years, average 25 years of age, in advance under the circadian rhythm model conditions (natural light, 16 h-light: 8 h-dark cycle, LD) for 1 weeks: room temperature 25 + 1 oC, 7:00, sleep time (no light period): 23:00 ~ dinner time: dinner time: supper time: The intensity of sleep time was 0.1 Lux; the subjects were free drinking water, smokeless alcohol hobby, daily activity and diet composition basically identical. Then the peripheral blood of the subjects was 6 ml every 4 h every day and night, the lymphocyte was separated, the total RNA was extracted and cDNA was extracted. The time point of day and night (zeitgeber time, ZT, total 6) was measured by the real-time fluorescence quantitative PCR method. The mRNA expression of the Clock and Bmal1 genes in the samples at each time point n=10) was verified by the dissolution curve and the gel electrophoresis strip. The rhythmic parameters were obtained by the cosine method and the Clock Lab software, and the circadian rhythm was analyzed by the amplitude test.
Result:
1. Under LD (16:8) light, the transcription of Clock and Bmal1 mRNA in normal adult peripheral blood lymphocytes showed obvious circadian oscillation (amplitude F test, P 0.05).
The peak phase of the 2. Clock gene is -161.70 + 17.36, the amplitude of the expression is 3.08 + 1.38, the median value is 11.70 + 1.58, the peak and the valley time are at ZT13 and ZT1 respectively, and the peak phase and the valley value mRNA level are 14.77 + 1.26 and 8.63 + 2.67, respectively.
The peak phase of the 3. Bmal1 gene is -177.55 + 23.48, the amplitude of the expression is 2.67 + 1.23, the median value is 5.50 + 1.52, the peak and the valley time are at ZT12 and ZT24 respectively, and the peak phase and the valley value mRNA level are 8.16 + 2.67 and 2.83 + 0.71, respectively.
In 4. LD (16:8) light, the expression level of Bmal1 gene in human peripheral blood lymphocytes at different day and night time was lower than that of Clock gene (P0.05). The peak phase, amplitude, peak time and valley time of the two gene transcription were the same (P0.05), while the level of the median of Bmal1 gene transcription, the mRNA level of the peak phase and the mRNA level of the valley value were reduced. (P0.05).
Conclusion:
Under 1. LD (16:8) light, the transcriptional oscillation of adult peripheral blood lymphocyte clocks gene Clock and Bmal1 has obvious circadian rhythms. The peak and valley time of Clock gene transcription are located at ZT13 and ZT1 respectively. The peak and valley time of Bmal1 gene are at ZT12 and ZT24 respectively.
In 2. LD (16:8) light, the peak phase, amplitude, peak time and valley time of the circadian clock genes of human peripheral blood lymphocyte clocks gene Clock and Bmal1 were the same as the valley time, and the two circadian rhythmic transcriptional characteristics were present.
Under 3. LD (16:8) light, the expression level of Bmal1 gene in human peripheral blood lymphocytes at different day and night time, the median value, the mRNA level of the peak phase and the mRNA level of the valley value phase were lower than those of the Clock gene.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R33
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