本文選題：Clock基因 + Bmal1基因��； 參考：《蘇州大學(xué)》2011年碩士論文

【摘要】：人和動物的生理機(jī)能、生化代謝、行為表現(xiàn)等常以24 h為周期發(fā)生規(guī)律性變動,這種變化的節(jié)律稱為晝夜節(jié)律。產(chǎn)生晝夜節(jié)律的結(jié)構(gòu)基礎(chǔ)是分子計(jì)時器—晝夜節(jié)律生物鐘(circadian clock)。對于主導(dǎo)節(jié)律調(diào)節(jié)的一些重要基因,稱之為生物鐘基因。機(jī)體的各種內(nèi)在晝夜節(jié)律性變化可在外界環(huán)境因素的作用下得到重設(shè)性調(diào)節(jié),以使機(jī)體活動與環(huán)境變化達(dá)成和諧統(tǒng)一。 引發(fā)和調(diào)控晝夜節(jié)律生物鐘的分子機(jī)制是在鐘輸入信號(如光照、溫度等)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)鐘信號的精確輸出。其中作為環(huán)路中正向調(diào)節(jié)成分的核心鐘基因Clock和Bmal1,已被證實(shí)不但24h節(jié)律性表達(dá)于中樞鐘組織如視交叉上核與松果體,而且也表達(dá)于包括血細(xì)胞在內(nèi)的各種外周鐘組織。上述基因產(chǎn)物以雜二聚體形式形成正向轉(zhuǎn)錄因子,與下游鐘基因Per(Period)、Cry(Cryptochromes)或tim (Timeless)啟動子區(qū)的E-box元件結(jié)合,激活一系列基因的轉(zhuǎn)錄和翻譯,因此,Clock和Bmal1在生物鐘的分子振蕩機(jī)制中被認(rèn)為起著極其重要的作用。然而,有關(guān)人外周血淋巴細(xì)胞核心鐘基因Clock和Bmal1的晝夜節(jié)律性轉(zhuǎn)錄在南極大陸特殊環(huán)境下有著怎樣的表現(xiàn),至今未明,這正是本文所要探討的內(nèi)容。 目的 探討赴南極前后科考隊(duì)員外周血淋巴細(xì)胞核心鐘基因Clock和Bmal1的晝夜節(jié)律性表達(dá)規(guī)律,旨在解析、拓展特殊環(huán)境下人類外周免疫鐘運(yùn)行的分子調(diào)控機(jī)制。 方法 8名健康、男性中國南極科考隊(duì)員志愿者,年齡24～30歲,平均25歲。赴南極前在晝夜節(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,起床時間:7:00,睡眠時間(無光照期):23:00～7:00,早餐時間:7:30～8:00,午餐時間:11:30～12:00,晚餐時間:5:30～6:00;睡眠時光照強(qiáng)度0.1 Lux;受試者自由飲水,無煙酒嗜好,在過去6個月內(nèi)未做過跨時區(qū)旅行和未接受過藥物治療,日�；顒雍惋嬍吵煞只疽恢�。隨后在一晝夜內(nèi),隨機(jī)選擇2位受試者,每隔4 h抽取各自外周血6 ml,血樣采集在4個不連續(xù)的隨機(jī)晝夜內(nèi)完成。分離其淋巴細(xì)胞,提取總RNA,逆轉(zhuǎn)錄為cDNA,采用實(shí)時熒光定量PCR方法,測定不同晝夜時點(diǎn)(zeitgeber time,ZT,共6個,每個時點(diǎn)n=8)每個樣品中Clock和Bmal1基因的mRNA表達(dá)量,并以熔解曲線和凝膠電泳條帶加以驗(yàn)證,通過余弦法和Clock Lab軟件獲取節(jié)律參數(shù),并經(jīng)振幅F檢驗(yàn)分析是否存在晝夜節(jié)律性表達(dá)。 選取相同8名男性科考隊(duì)員,赴南極后生活一年,經(jīng)歷了極晝與極夜光照差異、季節(jié)與氣候差異、氣溫與氣壓差異等自然環(huán)境變化。其晝夜模式生活條件、采血方式、取樣時間、實(shí)驗(yàn)檢測方法及數(shù)據(jù)統(tǒng)計(jì)學(xué)處理同上。 結(jié)果 1.科考隊(duì)員赴南極前外周血淋巴細(xì)胞核心鐘基因Clock和Bmal1的晝夜表達(dá) 1.1在LD(16:8)光制下,科考隊(duì)員外周血淋巴細(xì)胞鐘基因Clock和Bmal1的mRNA表達(dá)呈現(xiàn)明顯的晝夜節(jié)律性振蕩(振幅F檢驗(yàn),P0.05); 1.2 Clock基因的峰值相位-335.85±13.80,表達(dá)振幅3.46±1.27,中值-11.40±1.59,峰時和谷時分別位于ZT22和ZT10,峰時與谷時mRNA水平分別為-7.95±2.55和-14.85±1.32; 1.3 Bmal1基因的峰值相位-307.12±108.17,表達(dá)振幅2.77±1.11 ,中值-5.50±1.32,峰時和谷時分別位于ZT20和ZT8,峰時與谷時mRNA水平分別為-2.74±0.71和-8.25±2.33; 1.4 Clock和Bmal1晝夜節(jié)律性轉(zhuǎn)錄的比較:兩個基因LD(16:8)光制下在所檢測的6個晝夜時點(diǎn)中表達(dá)水平均有明顯差異(P0.05),Clock基因的表達(dá)水平較Bmal1基因降低;從晝夜節(jié)律性參數(shù)比較看出,兩個基因表達(dá)的峰值相位、振幅無差異(P0.05),而Clock基因轉(zhuǎn)錄的中值水平以及峰時mRNA水平和谷時mRNA水平均降低(P0.05),Clock峰時和谷時比Bmal1推后約2小時(P0.05)。 2.科考隊(duì)員赴南極后外周血淋巴細(xì)胞核心鐘基因Clock和Bmal1的晝夜表達(dá) 2.1八名科考隊(duì)員中分別有2人鐘基因Clock、3人Bmal1的轉(zhuǎn)錄表達(dá)具有明顯的晝夜節(jié)律性(振幅F檢驗(yàn),P0.05); 2.2二人Clock基因的峰值相位-42.28±5.27,表達(dá)振幅0.79±0.29,中值-9.02±0.24,峰時和谷時分別位于ZT3和ZT15,峰時與谷時mRNA水平分別為-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,表達(dá)振幅1.04±0.65,中值-8.50±0.45,峰時和谷時分別位于ZT12和ZT24,峰時與谷時mRNA水平分別為-7.46±0.41和-9.54±1.04; 2.4 Clock和Bmal1晝夜節(jié)律性轉(zhuǎn)錄的比較:存在晝夜節(jié)律性表達(dá)的兩個基因[Clock (n=2)和Bmal1 (n=3)]在所檢測的6個晝夜時點(diǎn)(除了ZT14)的表達(dá)水平及其晝夜節(jié)律性參數(shù)變化均無顯著性差異(P0.05); 2.5其他隊(duì)員的上述兩個基因不表現(xiàn)明顯的晝夜節(jié)律性轉(zhuǎn)錄特征(振幅F檢驗(yàn),P0.05)。這些無節(jié)律表達(dá)隊(duì)員的Clock基因,除晝夜時點(diǎn)ZT22以外,在其它各時點(diǎn)的表達(dá)量均低于Bmal1基因(P0.05)。 3.科考隊(duì)員赴南極前、后外周血淋巴細(xì)胞核心鐘基因Clock和Bmal1晝夜節(jié)律性轉(zhuǎn)錄的比較 3.1赴南極前、后鐘基因Clock晝夜節(jié)律性表達(dá)的比較:赴南極后的Clock晝夜節(jié)律性表達(dá),與赴南極前相比,除晝夜時點(diǎn)ZT18和ZT22以外,在其它時點(diǎn)的表達(dá)量均上調(diào)(P0.05)。隊(duì)員赴南極后Clock基因的節(jié)律性表達(dá)振幅減小、中值增大、谷時mRNA水平增大(P0.05),峰時和谷時分別推遲約5個小時。 3.2赴南極前、后鐘基因Bmal1晝夜節(jié)律性表達(dá)的比較:赴南極后的Bmal1晝夜節(jié)律性表達(dá),與赴南極前相比,除晝夜時點(diǎn)ZT10和ZT14以外,在其它時點(diǎn)的表達(dá)量均下調(diào)(P0.05)。隊(duì)員赴南極后Bmal1基因的節(jié)律性表達(dá)峰值相位推后,振幅、中值和峰時mRNA水平均下降(P0.05),峰時和谷時分別推遲約16個小時。 結(jié)論 1.赴南極前,在LD(16:8)自然光制下,科考隊(duì)員外周血淋巴細(xì)胞核心生物鐘基因Clock和Bmal1的轉(zhuǎn)錄具有明顯的晝夜節(jié)律性振蕩特征;Clock轉(zhuǎn)錄的峰時和谷時分別位于ZT22和ZT10,Bmal1的峰時和谷時分別位于ZT20和ZT8;Clock基因各晝夜時點(diǎn)的表達(dá)水平、中值以及峰時mRNA水平和谷時mRNA水平較Bmal1基因的降低,Clock峰時和谷時比Bmal1推后約2小時。 2.赴南極后,經(jīng)歷各種自然環(huán)境變化,相同8名隊(duì)員中多數(shù)人上述兩個基因不再表現(xiàn)明顯的晝夜節(jié)律性轉(zhuǎn)錄特征,Clock基因除晝夜時點(diǎn)ZT22以外,在其它各時點(diǎn)的表達(dá)量均低于Bmal1基因。 3.赴南極后,2人Clock基因、3人Bmal1基因的轉(zhuǎn)錄繼續(xù)維持明顯的晝夜節(jié)律性表達(dá),其中Clock轉(zhuǎn)錄的峰時和谷時分別位于ZT3和ZT15,Bmal1的峰時和谷時分別位于ZT12和ZT24;兩個基因在所檢測的6個晝夜時點(diǎn)(ZT14除外)的表達(dá)水平及其晝夜節(jié)律性參數(shù)的相應(yīng)變化基本一致。 4.與赴南極前相比,赴南極后的Clock晝夜節(jié)律性表達(dá),除晝夜時點(diǎn)ZT18和ZT22以外,在其它時點(diǎn)均為上調(diào),節(jié)律振幅減小、中值增大、谷時mRNA水平增大,節(jié)律峰時和谷時分別推遲約5個小時。 5.與赴南極前相比,赴南極后的Bmal1晝夜節(jié)律性表達(dá),除晝夜時點(diǎn)ZT10和ZT14以外,在其它時點(diǎn)均為下調(diào),節(jié)律峰值相位推后,振幅、中值、峰時mRNA水平均下降,節(jié)律峰時和谷時分別推遲約16個小時。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R33

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