本文選題：β-arrestin-TRAF6相互作用 + TLR-IL-1R信號通路��； 參考：《中國科學(xué)院研究生院（上海生命科學(xué)研究院）》2007年碩士論文

【摘要】： Toll樣受體-白介素1受體(TLR-IL-1R)信號轉(zhuǎn)導(dǎo)在宿主的免疫應(yīng)答和抵抗病原入侵方面發(fā)揮著極為重要的作用。TLR-IL-1R信號的過度激活會導(dǎo)致機(jī)體產(chǎn)生一些自身免疫性疾病。因此TLR-IL-1R信號必須被精細(xì)調(diào)控,研究機(jī)體對該信號的調(diào)控機(jī)制有助于我們更好地認(rèn)識人體的免疫調(diào)控,從而有效防治自身免疫疾病等傷害。 β抑制蛋白(β-arrestin)是一類多功能的蛋白,其經(jīng)典功能是作為接頭蛋白介導(dǎo)G蛋白偶聯(lián)受體(GPCR)的脫敏和內(nèi)吞。近年來的研究顯示,β-arrestin還參與GPCR和其他信號通路間的通訊或直接調(diào)控其他信號的轉(zhuǎn)導(dǎo),如β-arrestin介導(dǎo)β2AR信號對免疫系統(tǒng)的調(diào)節(jié)作用。但β-arrestin與TLR-IL-1R信號間的關(guān)系則未見報(bào)道。 我們的研究發(fā)現(xiàn),β-arrestin可以直接結(jié)合TLR-IL-1R下游關(guān)鍵分子TRAF6,抑制NF-κB的激活,從而抑制TLR-IL-1R下游多種細(xì)胞因子的轉(zhuǎn)錄和表達(dá)。在脂多糖(LPS)刺激下,與對照野生型小鼠成纖維細(xì)胞(MEF)比較,β-arrestin1/β-arrestin2雙敲除小鼠成纖維細(xì)胞中Ccl2、TNFα和IκBδ等細(xì)胞因子的轉(zhuǎn)錄水平明顯升高。將β-arrestin1或β-arrestin2重新轉(zhuǎn)回雙敲除的MEF細(xì)胞時,LPS刺激導(dǎo)致Ccl2、TNFα和IκBδ轉(zhuǎn)錄水平的升高則受到明顯抑制。此外,在Hela細(xì)胞中過表達(dá)β-arrestin1或β-arrestin2也都能抑制IL-1β刺激下Ccl2、IL-6和IL-8轉(zhuǎn)錄水平的升高。進(jìn)一步檢測蛋白表達(dá)水平發(fā)現(xiàn),在THP-1細(xì)胞中用RNAi的方法降低內(nèi)源β-arrestin1的表達(dá)水平可以增強(qiáng)THP-1細(xì)胞在LPS刺激下的細(xì)胞因子表達(dá)。這些研究從細(xì)胞水平上證明β-arrestin結(jié)合TRAF6可以負(fù)調(diào)控TLR-IL-1R信號通路,抑制TLR-IL-1R激活導(dǎo)致的下游細(xì)胞因子表達(dá)。接下來,我們通過腹腔注射LPS建立內(nèi)毒素刺激小鼠模型的研究發(fā)現(xiàn),注射LPS情況下,β-arrestin2基因敲除小鼠比對照野生型小鼠產(chǎn)生更多的Ccl2, TNFα, IL-1β, IL-6和IκBδ等,進(jìn)一步從動物水平證實(shí)了β-arrestin是TLR-IL-1R信號的一個負(fù)調(diào)控因子的結(jié)論。 Arrestin基因家族在進(jìn)化上具有高度的保守性,分為有器官特異性分布的Visual arrestin基因家族和普遍分布的β-arrestin基因家族。對于β-arrestin基因家族的研究長期集中在哺乳動物中。最近,有文獻(xiàn)報(bào)道了斑馬魚β-arrestin2基因在胚胎發(fā)育過程中參與了smoothen信號通路。但通過文獻(xiàn)檢索和序列查找,我們至今沒有發(fā)現(xiàn)低等非哺乳類脊椎動物中關(guān)于β-arrestin1基因的報(bào)道。 本研究通過Genebank中一小段高度相似序列,使用RACE方法第一次從斑馬魚中克隆獲得了β-arrestin1基因,并通過多序列比對和進(jìn)化分析,定位了該基因上具有的β-arrestin家族保守的Clathrin結(jié)合域,AP-2和MAPK結(jié)合位點(diǎn)。激光共聚交熒光顯微鏡的結(jié)果顯示,外源表達(dá)斑馬魚GFP-β-arrestin1蛋白在細(xì)胞質(zhì)和細(xì)胞核內(nèi)均有分布,這和已知的β-arrestin1蛋白亞細(xì)胞定位一致。進(jìn)一步的β2AR受體內(nèi)吞實(shí)驗(yàn)則在功能上驗(yàn)證了新克隆的基因能夠參與GPCR信號轉(zhuǎn)導(dǎo),具有β-arrestin家族保守的功能。此外,實(shí)時定量RT-PCR的結(jié)果顯示,斑馬魚β-arrestin1和β-arrestin2基因在發(fā)育過程中都存在時序表達(dá)的現(xiàn)象。這提示了斑馬魚β-arrestin可能在胚胎發(fā)育過程中具有重要的生物學(xué)功能。因此,本文報(bào)道了第一個被克隆的非哺乳類脊椎動物中的β-arrestin1基因,并從序列、蛋白亞細(xì)胞定位以及功能上驗(yàn)證了該基因的進(jìn)化地位,提示了該基因潛在的生物學(xué)功能。
[Abstract]:The Toll like receptor - IL-1 receptor (TLR-IL-1R) signal plays an extremely important role in the host's immune response and resistance to pathogenic invasion. The overactivation of.TLR-IL-1R signals will lead to some autoimmune diseases. Therefore, the TLR-IL-1R signal must be carefully regulated, and the mechanism of the body's regulation of the signal is studied. It helps us to better understand the immune regulation of human body, so as to effectively prevent and control autoimmune diseases.
Beta suppressor (beta -arrestin) is a kind of multifunctional protein whose classic function is to mediate the desensitization and endocytosis of G protein coupled receptor (GPCR) as a joint protein. Recent studies have shown that beta -arrestin also participates in the communication between GPCR and other signaling pathways or directly regulates the transduction of his signal, such as beta -arrestin mediated beta 2AR signal to immunity The regulatory role of the epidemic system. However, the relationship between beta -arrestin and TLR-IL-1R signal has not been reported.
Our study found that beta -arrestin can directly bind to the key molecule TRAF6 of downstream TLR-IL-1R, inhibit the activation of NF- kappa B, and inhibit the transcription and expression of a variety of cytokines in the downstream of TLR-IL-1R. Under the stimulation of lipopolysaccharide (LPS), the beta -arrestin1/ beta -arrestin2 double knockout in mice is compared with that of the control wild type mouse fibroblasts (MEF). The transcriptional level of cell factors such as Ccl2, TNF A and I kappa B Delta in the cells was significantly increased. When the beta or beta -arrestin2 was retransferred to the double knockout MEF cells, LPS stimulation led to Ccl2, TNF A and I kappa B delta transcriptional levels were significantly inhibited. Further detection of Ccl2, IL-6 and IL-8 transcriptional levels. Further detection of protein expression levels found that the expression level of endogenous beta -arrestin1 decreased by RNAi in THP-1 cells can enhance the expression of cytokines in THP-1 cells under LPS stimulation. These studies show that beta -arrestin binding TRAF6 can negatively regulate TLR-IL-1R TLR-IL-1R on the cell level. The signal pathway inhibited the expression of downstream cytokine induced by TLR-IL-1R activation. Next, we found that the mice injected with LPS by intraperitoneal injection of endotoxin stimulated mice model. In the case of LPS injection, the beta -arrestin2 knockout mice produced more Ccl2, TNF a, IL-1 beta, IL-6 and I kappa B Delta, and further from the animals. The level confirms that beta -arrestin is a negative regulator of TLR-IL-1R signal.
The Arrestin gene family is highly conserved in evolution, divided into the Visual arrestin gene family with an organ specific distribution and the widely distributed beta -arrestin family. The study of the beta -arrestin gene family has been concentrated in mammals for a long time. Recently, there have been reports of the development of the zebrafish beta -arrestin2 gene in embryo development. Smoothen signaling pathway is involved in the process, but we have not found a report on the gene of beta -arrestin1 in lower non mammalian vertebrates through literature retrieval and sequence search.
In this study, the beta -arrestin1 gene was obtained from zebrafish for the first time by RACE method, and the conservative Clathrin binding domain of the beta -arrestin family, the binding site of AP-2 and MAPK in the gene, and the laser copolymerization fluorescence microscope were obtained by using the method of multiple sequence alignment and evolutionary analysis in Genebank. The results showed that the GFP- beta -arrestin1 protein was distributed in both cytoplasm and nucleus of zebrafish, which was in accordance with the known subcellular localization of beta -arrestin1 protein. Further beta 2AR receptor endocytosis test demonstrated that the newly cloned gene could be involved in GPCR signal transduction and had the conserved function of the beta -arrestin family. In addition, the results of real-time quantitative RT-PCR show that the gene of zebrafish beta -arrestin1 and beta -arrestin2 have time series expression during development, which suggests that zebrafish beta -arrestin may have important biological functions during the development of embryo. Therefore, this paper reports the beta -a of the first cloned non mammalian vertebrate. The rrestin1 gene was verified by sequence, protein subcellular location and function, indicating the potential biological function of the gene.
【學(xué)位授予單位】：中國科學(xué)院研究生院（上海生命科學(xué)研究院）
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 孟安明;斑馬魚胚胎發(fā)育的功能染色體組[J];實(shí)驗(yàn)動物科學(xué)與管理;2003年S1期

2 本刊編輯部;暴露于內(nèi)分泌干擾物的斑馬魚的蛋白組學(xué)[J];環(huán)境與健康雜志;2004年02期

3 林云;李榮英;劉智;於惠敏;宋懷東;;斑馬魚甲狀腺球蛋白基因的克隆及其組織表達(dá)[J];中華內(nèi)分泌代謝雜志;2006年02期

4 高世榮;修瑞琴;郭琪;許永香;任改英;;硒對斑馬魚的毒性研究[J];中國公共衛(wèi)生學(xué)報(bào);1993年01期

5 趙寶全,董武,王思珍,方厚華,章金剛;17β-雌二醇對斑馬魚初期胚體節(jié)形成影響的研究[J];中國比較醫(yī)學(xué)雜志;2005年01期

6 楊雁;劉玉;孫衛(wèi)斌;;斑馬魚在牙齒發(fā)育研究中的應(yīng)用[J];口腔生物醫(yī)學(xué);2010年04期

7 汪曉華;;用鏈球菌-斑馬魚模型研究細(xì)菌致病機(jī)制[J];國外醫(yī)學(xué)(微生物學(xué)分冊);2003年01期

8 李潔斐,李衛(wèi)華,金泰^，

本文編號：2042344