成年人外周血淋巴細(xì)胞生物鐘基因Clock和Bmall晝夜表達(dá)規(guī)律的研究
發(fā)布時(shí)間：2018-08-03 11:01
【摘要】： 機(jī)體的生理功能、生化代謝、行為改變等均表現(xiàn)晝夜節(jié)律現(xiàn)象。這種節(jié)律的紊亂可導(dǎo)致多種疾病,如飛行跨越不同地理時(shí)區(qū)的時(shí)差反應(yīng)、晝夜輪班制作業(yè)引起的睡眠障礙、激素分泌異常誘發(fā)的代謝失調(diào)與提前衰老、特殊節(jié)律缺陷導(dǎo)致的神經(jīng)精神疾病等。揭示晝夜節(jié)律的發(fā)生原因和調(diào)節(jié)機(jī)制,對(duì)解釋正常機(jī)體的生理、生化和行為現(xiàn)象以及因晝夜節(jié)律紊亂而引發(fā)的病理生理過(guò)程,乃至提出相應(yīng)的防治措施等都有重要的理論意義和應(yīng)用價(jià)值。 調(diào)控晝夜節(jié)律的生物鐘包括中樞鐘組織視交叉上核(Suprachiasmatic nucleus, SCN)、松果體(Pineal gland, PG)和各種外周鐘組織。其分子機(jī)制是在鐘輸入信號(hào)的作用下,若干鐘基因、鐘相關(guān)基因和鐘控基因及其蛋白產(chǎn)物通過(guò)轉(zhuǎn)錄—翻譯—翻譯后事件的相互銜接,組成鐘振蕩器的自身調(diào)控反饋環(huán)路,實(shí)現(xiàn)鐘信號(hào)的精確輸出。作為重要的鐘基因,Clock和Bmal1主要表達(dá)于SCN、松果體和視網(wǎng)膜,可編碼具有堿性螺旋—環(huán)—螺旋(base helix-loop-helix, bHLH)以及PAS( PER-ASNT-SIM )結(jié)構(gòu)域家族的轉(zhuǎn)錄因子,互為配體的共同產(chǎn)物—CLOCK:BMAL1蛋白在細(xì)胞核內(nèi)形成雜二聚體,作為正向調(diào)節(jié)子與下游鐘基因Per(Period)、Cry(cryptochromes)或tim (timeless)啟動(dòng)子區(qū)的E-box元件結(jié)合,激活這些基因的轉(zhuǎn)錄和翻譯。 有關(guān)哺乳類(lèi)中樞不同部位鐘基因Clock和Bmal1晝夜表達(dá)規(guī)律的研究,已有報(bào)道。而外周鐘組織尤其是人類(lèi)外周淋巴細(xì)胞Clock和Bmal1的晝夜節(jié)律性表達(dá)及其光反應(yīng)性還不清楚,這正是本研究所要探討的內(nèi)容。 目的: 探討成年人外周血淋巴細(xì)胞鐘基因Clock和Bmal1的晝夜節(jié)律性定量表達(dá)規(guī)律,旨在進(jìn)一步解析外周免疫鐘運(yùn)行的分子調(diào)控機(jī)制。 方法: 10名健康男性志愿者,年齡24～30歲,平均25歲。預(yù)先在晝夜節(jié)律模式條件(自然光制,16 h-light : 8 h-dark cycle, LD)下生活1周:室溫25±1 oC,起床時(shí)間:7:00,睡眠時(shí)間(無(wú)光照期):23:00～7:00,早餐時(shí)間:7:30～8:00,午餐時(shí)間:11:30～12:00,晚餐時(shí)間:5:00～6:00;睡眠時(shí)光照強(qiáng)度0.1 Lux;受試者自由飲水,無(wú)煙酒嗜好,日�；顒�(dòng)和飲食成分基本一致。隨后在一晝夜內(nèi)每隔4 h抽取各受試者外周血6 ml,分離淋巴細(xì)胞,提取總RNA,逆轉(zhuǎn)錄為cDNA,采用實(shí)時(shí)熒光定量PCR方法,測(cè)定不同晝夜時(shí)點(diǎn)(zeitgeber time,ZT,共6個(gè),每個(gè)時(shí)點(diǎn)n=10)樣品中Clock和Bmal1基因的mRNA表達(dá)量,并以溶解曲線(xiàn)和凝膠電泳條帶加以驗(yàn)證,通過(guò)余弦法和Clock Lab軟件獲取節(jié)律參數(shù),并經(jīng)振幅檢驗(yàn)分析是否存在晝夜節(jié)律。 結(jié)果: 1. LD(16:8)光制下,正常成年人外周血淋巴細(xì)胞鐘基因Clock和Bmal1的mRNA轉(zhuǎn)錄呈現(xiàn)明顯的晝夜節(jié)律性振蕩(振幅F檢驗(yàn),P0.05); 2. Clock基因的峰值相位-161.70±17.36,表達(dá)振幅3.08±1.38,中值11.70±1.58,峰值和谷值時(shí)間分別位于ZT13和ZT1,峰值相與谷值相mRNA水平分別為14.77±1.26和8.63±2.67; 3. Bmal1基因的峰值相位-177.55±23.48,表達(dá)振幅2.67±1.23,中值5.50±1.52,峰值和谷值時(shí)間分別位于ZT12和ZT24,峰值相與谷值相mRNA水平分別為8.16±2.67和2.83±0.71; 4. LD(16:8)光制下,人外周血淋巴細(xì)胞Bmal1基因在不同晝夜時(shí)點(diǎn)的表達(dá)水平較Clock基因降低(P0.05),兩個(gè)基因轉(zhuǎn)錄的峰值相位、振幅、峰值時(shí)間和谷值時(shí)間相一致(P0.05),而B(niǎo)mal1基因轉(zhuǎn)錄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)。 結(jié)論: 1. LD(16:8)光制下,成年人外周血淋巴細(xì)胞生物鐘基因Clock和Bmal1的轉(zhuǎn)錄振蕩具有明顯的晝夜節(jié)律性,Clock基因轉(zhuǎn)錄的峰值和谷值時(shí)間分別位于ZT13和ZT1,Bmal1基因的峰值和谷值時(shí)間分別位于ZT12和ZT24; 2. LD(16:8)光制下,人外周血淋巴細(xì)胞生物鐘基因Clock和Bmal1晝夜節(jié)律性表達(dá)的峰值相位、振幅、峰值時(shí)間和谷值時(shí)間相一致,二者存在同步化的晝夜節(jié)律性轉(zhuǎn)錄特征; 3. LD(16:8)光制下,人外周血淋巴細(xì)胞Bmal1基因不同晝夜時(shí)點(diǎn)的表達(dá)水平、中值、以及峰值相的mRNA水平和谷值相的mRNA水平較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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R33
本文編號(hào)：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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R33
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