本文選題：CRH + CRHR1��； 參考：《浙江大學》2012年博士論文

【摘要】：低氧應(yīng)激是應(yīng)激的重要形式之一。為適應(yīng)低氧環(huán)境、進行自我保護,機體形成了在不同氧環(huán)境下的基因表達調(diào)控機制。促腎上腺皮質(zhì)激素釋放激素(Corticotropin-releasing hormone, CRH)家族在機體低氧應(yīng)激過程中通過調(diào)節(jié)下丘腦-垂體-腎上腺皮質(zhì)(hypothalamus pituitary adrenal, HPA)軸、神經(jīng)內(nèi)分泌系統(tǒng)、自主神經(jīng)系統(tǒng)和免疫系統(tǒng)功能,參與調(diào)節(jié)機體的低氧應(yīng)激過程,進而穩(wěn)定機體內(nèi)環(huán)境。促腎上腺皮質(zhì)激素釋放激素1型受體(Corticotropin releasing hormone receptor1, CRHR1)介導CRH生理作用的發(fā)揮,其基因表達調(diào)控是應(yīng)激誘導HPA軸反應(yīng)的關(guān)鍵環(huán)節(jié)。我們實驗室前期研究發(fā)現(xiàn)低氧應(yīng)激激活HPA軸,誘導大鼠等動物腦不同部位以及腺垂體CRHR1mRNA表達上調(diào),提示低氧下轉(zhuǎn)錄因子可能通過與其作用元件結(jié)合,在轉(zhuǎn)錄水平調(diào)控CRHR1mRNA的表達。本研究旨在探討低氧應(yīng)激下NF-κB和AP-1參與CRHR1轉(zhuǎn)錄調(diào)節(jié)的機制。研究方法 將大鼠垂體CRHR1基因5’調(diào)控區(qū)-2161～+347段不同長度DNA序列亞克隆入不含啟動子的螢光素酶載體pGL3中,構(gòu)建各種質(zhì)粒,轉(zhuǎn)染AtT20細胞,檢測其活性與內(nèi)參pRL-TK螢光素酶活性比值；利用整體間歇低氧應(yīng)激動物模型,研究低氧誘導大鼠垂體CRHR1基因表達變化；應(yīng)用EMSA方法檢測轉(zhuǎn)錄因子在體外與靶序列結(jié)合活性；應(yīng)用定量RealtimePCR方法檢測整體水平CRHR1mRNA的表達。研究結(jié)果如下： 1.獲得大鼠垂體CRHR1基因5’調(diào)控區(qū)-2161～+347段DNA序列,全長為2508bp,經(jīng)過生物信息學分析,發(fā)現(xiàn)該序列分別含有5個c-jun/AP-1,3個GR,4個NF-κB以及8個HIF-1結(jié)合位點。 2.成功構(gòu)建質(zhì)粒p2161-18T和p2161Luc,分別用于測序和檢測CRHR1基因5’端調(diào)控區(qū)轉(zhuǎn)錄活性。根據(jù)轉(zhuǎn)錄因子結(jié)合元件在CRHR1基因5’調(diào)控區(qū)-2161～+347段的分布,又分別構(gòu)建了p1833Luc、p1795Luc、p1692Luc、p1289Luc、p1248Luc、 p1218Luc、p1140Luc、p838Luc、p687Luc、p360Luc等系列缺失體質(zhì)粒。進一步構(gòu)建了含-809～-800位點的NF-κB結(jié)合元件的突變體質(zhì)粒p838mLuc和含有-1277～1271位點的c-jun/AP-1結(jié)合元件的突變體質(zhì)粒p1289mLuc和以及c-jun的真核表達質(zhì)粒pcDNA3.1-c-jun. 3.發(fā)現(xiàn)p2161Luc、p1289Luc、p838Luc在AtT20細胞中的活性隨低氧時間延長而呈現(xiàn)增強的趨勢,在24小時達到最大值。P2161Luc和p1289Luc的活性在低氧暴露8-12小時期間曲線平緩。 4.發(fā)現(xiàn)低氧暴露24小時,p838Luc在AtT20細胞中的活性與常氧對照組相比顯著增強,這一現(xiàn)象可以被NF-κB抑制劑(PDTC)抑制；而突變體p838mLuc活性無明顯增強。大鼠垂體CRHR1基因5’調(diào)控區(qū)-809～-800區(qū)域的結(jié)合元件在體外能與AtT20細胞以及大鼠垂體的核蛋白中的NF-κB結(jié)合,且低氧進一步增強二者結(jié)合活性,此結(jié)合可以被PDTC、p65抗體、100倍濃度的冷探針消除；突變探針(-809～-800)不再與NF-κB結(jié)合。在整體低壓艙模擬5km間歇低氧(10.8%02,4h/d)條件下,CRHR1mRNA表達表現(xiàn)為一個先下降后增強的過程,在第2天表達顯著下降,第5天表達上升,二者均可被PDTC反轉(zhuǎn)；7km(7.8%02)連續(xù)低氧8h,CRHR1mRNA表達下降幅度更大,此結(jié)果同樣可被PDTC反轉(zhuǎn)。 5.發(fā)現(xiàn)大鼠垂體CRHR1基因5’調(diào)控區(qū)-1277～1271區(qū)域的結(jié)合元件在體外能與AtT20細胞核蛋白中的c-jun/AP-1結(jié)合,低氧暴露進一步增強二者結(jié)合活性,此結(jié)合可以被c-jun磷酸化抗體、100倍濃度的冷探針消除。突變探針(-1277～1271)不再與c-jun/AP-1結(jié)合。p1289Luc轉(zhuǎn)染AtT20細胞,低氧暴露24h后,CRHR1基因5’調(diào)控區(qū)-1289～+347段的轉(zhuǎn)錄活性較常氧對照組增幅達到最大,此增幅可以被2μg pcDNA3.1-c-jun的共表達完全抑制。 結(jié)論 1CRFR1基因5’調(diào)控區(qū)-2161～+347參與低氧上調(diào)CRFR1基因轉(zhuǎn)錄。 2.證明了大鼠垂體CRHR1基因5’調(diào)控區(qū)-809～-800位點的NF-κB結(jié)合元件促進低氧誘導的大鼠垂體CRHR1基因的轉(zhuǎn)錄。 3.大鼠垂體CRHR1基因5’調(diào)控區(qū)-1277～1271位點的結(jié)合元件與AP-1在低氧暴露下結(jié)合活性增強,在AtT20細胞低氧暴露8～12小時AP-1可能參與抑制CRHR1基因的轉(zhuǎn)錄活性。
[Abstract]:Hypoxia stress is one of the important forms of stress. In order to adapt to the hypoxic environment, self protection, the body forms the regulation mechanism of gene expression in different oxygen environment. The Corticotropin-releasing hormone, CRH family regulates the hypothalamus pituitary adrenal cortex in the process of hypoxic stress. Hypothalamus pituitary adrenal (HPA) axis, neuroendocrine system, autonomic nervous system and immune system function, participate in regulating the body's hypoxia stress process, and then stabilize the body environment. Adrenocorticotropin releasing hormone 1 receptor (Corticotropin releasing hormone receptor1, CRHR1) mediates the physiological role of CRH. The regulation of gene expression is the key link in stress induced HPA axis response. In our previous laboratory, we found that hypoxia stress activates the HPA axis and induces the up regulation of CRHR1mRNA expression in different parts of the brain and adenohypophysis in rats, suggesting that the hypoxic transcription factors may be combined with its components and regulate the table of CRHR1mRNA at the transcriptional level. The aim of this study is to explore the mechanism of NF- kappa B and AP-1 involved in CRHR1 transcriptional regulation under hypoxia stress.
The -2161 ~ +347 segment of the rat pituitary CRHR1 gene 5 'gene was subcloned into the fluoro enzyme carrier pGL3 without promoter, and various plasmids were constructed and transfected to AtT20 cells to detect the activity ratio of the activity to the internal parameter pRL-TK luciferase activity, and the hypoxic induced rat pituitary C was studied by using the whole intermittent hypoxia stress animal model. RHR1 gene expression changes; EMSA method was used to detect the binding activity of transcription factors to target sequences in vitro; quantitative RealtimePCR method was used to detect the overall level of CRHR1mRNA expression. The results were as follows:
1. the DNA sequence of -2161 to +347 segment of the rat pituitary CRHR1 gene was obtained. The total length of the DNA sequence was 2508bp. Through bioinformatics analysis, it was found that the sequence contained 5 c-jun/AP-1,3 GR, 4 NF- kappa B and 8 HIF-1 binding sites.
2. the plasmid p2161-18T and p2161Luc were successfully constructed and used to sequence and detect the transcriptional activity of the 5 'terminal regulatory region of the CRHR1 gene, respectively. According to the distribution of the transcription factor binding element in the -2161 to +347 segment of the CRHR1 gene 5' regulatory region, the p1833Luc, p1795Luc, p1692Luc, p1289Luc, and p1248Luc were constructed, respectively. A series of missing body plasmids. The mutant body p838mLuc of the NF- kappa B binding element containing -809 to -800 and the c-jun/AP-1 binding element containing -1277 to 1271, and the eukaryotic expression plasmids pcDNA3.1-c-jun. of c-jun, are constructed.
3. it was found that the activity of p2161Luc, p1289Luc and p838Luc in AtT20 cells increased with the prolongation of hypoxia time, and the maximum value of.P2161Luc and p1289Luc at 24 hours was slow during 8-12 hours of hypoxia exposure.
4. after 24 hours of hypoxia exposure, the activity of p838Luc in AtT20 cells was significantly enhanced compared with that of the normal oxygen control group. This phenomenon could be inhibited by NF- kappa B inhibitor (PDTC), but the activity of the mutant p838mLuc was not significantly enhanced. The binding element in the -809 to -800 region of the pituitary CRHR1 gene 5 'regulation region could be in vitro with AtT20 cells and rats. The combination of NF- kappa B in the nuclear protein of the pituitary, and the hypoxia further strengthens the two binding activity, which can be eliminated by the cold probe of PDTC, p65, and 100 times; the mutant probe (-809 to -800) is no longer combined with NF- kappa B. Under the condition of the analog 5km intermittent hypoxia (10.8% 02,4h/d) in the whole hypobaric chamber, the CRHR1mRNA expression is a first descending. The expression of the enhanced process decreased significantly at second days, the fifth day expression rose and the two could be reversed by PDTC; 7km (7.8%02) was a continuous hypoxic 8h, and the CRHR1mRNA expression decreased significantly, and this result could also be reversed by PDTC.
5. it was found that the binding element in the -1277 ~ 1271 region of the CRHR1 gene 5 'region of the rat pituitary could be combined with the c-jun/AP-1 in the AtT20 nuclear protein in vitro, and the hypoxia exposure further enhanced the two binding activity, which could be eliminated by the c-Jun phosphorylated antibody and the cold probe at 100 times the concentration. The mutant probe (-1277 ~ 1271) was no longer with the c-jun/AP-1 junction. After transfection of.P1289Luc to AtT20 cells, after hypoxia exposure to 24h, the transcription activity of -1289 to +347 segment of the CRHR1 gene 5 'regulatory region was the largest, and the increase could be completely suppressed by co expression of 2 mu g pcDNA3.1-c-jun.
conclusion
1CRFR1 gene 5 'regulatory region -2161 ~ +347 participates in hypoxia and upregulates CRFR1 gene transcription.
2. it is proved that the NF- kappa B binding element of the -809 to -800 locus in the 5 'regulatory region of the pituitary CRHR1 gene promotes the transcription of CRHR1 gene in rat pituitary induced by hypoxia.
The binding element of the -1277 ~ 1271 loci of the pituitary CRHR1 gene 5 'regulatory region of the 3. rats was enhanced by the binding activity of AP-1 under hypoxia exposure, and AP-1 may be involved in inhibiting the transcriptional activity of the CRHR1 gene in the AtT20 cell hypoxia exposure for 8~12 hours.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R346

【參考文獻】

相關(guān)期刊論文 前1條

1 陳志,杜繼曾;低氧對下丘腦促腎上腺皮質(zhì)素－釋放激素和前垂體cAMP的作用[J];Acta Pharmacologica Sinica;1996年06期

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本文編號：1908846