本文關(guān)鍵詞： TGFβ 肝臟 酵母雙雜交 蛋白相互作用　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2007年博士論文　論文類型：學(xué)位論文

【摘要】： TGFβ即轉(zhuǎn)化生長因子(transforming growth factor)，，是一類多功能的細(xì)胞因子，其信號途徑及相關(guān)功能也是近年來的研究熱點。以往的研究表明，TGFβ與細(xì)胞的增殖、分化、遷移及凋亡有關(guān)；在機體中參與了從炎癥反應(yīng)、組織修復(fù)到骨骼形成、胚胎發(fā)育等一系列重要的生物學(xué)過程；并與某些人類疾病的發(fā)生密切相關(guān)。特別是在肝臟中，該信號通路的異常與肝炎、肝纖維化、肝硬化以及肝癌都有密切關(guān)系。因此對于該通路的研究具有十分重要的生物學(xué)意義；而對其各組分相互作用蛋白的研究將是闡明其功能及調(diào)控機制的關(guān)鍵所在。 本文采用高通量規(guī)�；牡鞍踪|(zhì)相互作用研究方法Y2H技術(shù)，以TGFβ信號通路中的重要分了Smad2、3、4為誘餌，在正常成人肝臟cDNA文庫中進行相互作用蛋白的篩選。三個誘餌分子的篩選中，共獲得125個陽性克隆。經(jīng)過測序比對，排除非編碼序列及不符合閱讀框的序列后，得到二個誘餌的87個正確獵物克隆，匹配上32個基因共包含34個相互作用對。經(jīng)文獻調(diào)研，其中6對為已知的相互作用，剩余28對即為本研究新發(fā)現(xiàn)的相互作用。 酵母回轉(zhuǎn)驗證中97％(33/34)的相互作用仍表現(xiàn)為陽性；隨機選取10對相互作用進行CO-IP，陽性率達到了100％，表明相互作用的可信度較高。采用生物信息學(xué)的方法，結(jié)合基因敲除信息對得到的相互作用進行了生物學(xué)相關(guān)性分析。結(jié)果顯示：66％的相互作用中獵物和誘餌具有相同的基因敲除表型；26％的獵物和誘餌具有基因共表達特性；26％的相互作用蛋白共享到GO中第六級功能注釋；56％的相互作用從網(wǎng)絡(luò)拓?fù)浣Y(jié)構(gòu)學(xué)分析具有較高的可信度。最后整合相關(guān)參數(shù)的評分結(jié)果，對每一對相互作用給出總體評分：53％(18/34)的相互作用具有高可信度。 最后，將得到的相互作用以及整合了已知相互作用的數(shù)據(jù)進行可視化分析，構(gòu)建相互作用網(wǎng)絡(luò)圖，對篩選結(jié)果進行補充和拓展。結(jié)合報告基因?qū)嶒灲Y(jié)果，我們推測篩選到的Smads相互作用蛋白在TGFβ信號通路中可能具有重要的調(diào)控作用。USP7：去泛素化酶，調(diào)控Smads蛋白在細(xì)胞中的表達水平；PPP1R12C：一種新的Smads蛋白去磷酸化途徑，參與TGFβ信號的活性調(diào)節(jié)；SQSTM1：作為支架蛋白通過介導(dǎo)Smads與其它蛋白的相互作用發(fā)揮對該信號通路的調(diào)控作用；此外本文篩選到Smad4與NLK的相互作用，這提示W(wǎng)nt/TGFβ信號通路可能具有新的交互聯(lián)絡(luò)方式。 另外，本文對Smad3復(fù)合體的研究進行了初步探索，擬采用串聯(lián)親和純化的方法在肝臟相關(guān)細(xì)胞系中分離純化Smad3復(fù)合體進行質(zhì)譜鑒定。首先構(gòu)建了靶基因的逆轉(zhuǎn)錄病毒載體，轉(zhuǎn)染包裝細(xì)胞后進行了陽性克隆的篩選，結(jié)果待鑒定。 綜上所述，本文利用Y2H技術(shù)對TGF信號通路中的重要組分進行了相互作用蛋白的研究。通過其他方法的相互作用驗證、生物信息學(xué)分析以及初步的功能實驗初探，推測一些新篩選到的相互作用蛋白在TGFβ信號通路中可能具有重要的調(diào)控作用，為探索該通路發(fā)揮功能的新的作用機制提供了線索。
[Abstract]:TGF is the transforming growth factor beta (transforming growth factor), is a kind of multifunctional cytokine, its signal pathway and related functions is the hotspot in recent years. Previous studies showed that TGF and beta cell proliferation, differentiation, migration and apoptosis; in the body is involved in inflammation and tissue repair to bone formation, embryonic development and a series of important biological processes; and is closely related to the occurrence of certain human diseases. Especially in the liver, liver fibrosis and the abnormal signal pathway, hepatitis, cirrhosis and liver cancer are closely related. So it has very important biological significance for the study of the pathway of the study; the protein interactions in each group will be the key to clarify the function and regulatory mechanism.
This paper uses high-throughput scale protein interaction research methods of Y2H technology, the TGF beta signaling pathway plays an important role in the Smad2,3,4 as bait, screening interaction protein in normal human liver cDNA library. The three screening baits, 125 positive clones were obtained. After sequencing, the exclusion of non encoding sequence and does not meet the reading frame sequence, two bait 87 correct prey, cloning, the interaction of 34. Contains a total of 32 genes on the literature research, of which 6 of the known interaction, the remaining 28 of the interaction of the new findings.
In 97% yeast rotary verification (33/34) interaction is shown to be positive; the interaction of 10 randomly selected for CO-IP, the positive rate reached 100%, showed high reliability of the interaction. By using the method of bioinformatics, combined with gene knockout on the information interaction of biological correlation analysis. The results showed the prey and bait: interaction in 66% with the same gene knockout phenotype; 26% with characteristics of prey and bait gene co expression; interacting protein 26% share to sixth GO in 56% functional annotation; the interaction in the network topological analysis has higher reliability. Finally the integration parameters related to the scoring results for each of the interaction, presented the total score: 53% (18/34) interaction with high confidence.
Finally, the interaction and integration of the known interaction data visualization analysis, construct the interaction network diagram, supplement and extension of the screening results. Combined with the reporter gene assay results, we speculate that the screening of the proteins interacting with Smads may play an important role for.USP7 in regulation of TGF beta signaling pathway to ubiquitin enzyme, regulating the expression levels of Smads protein in cells; PPP1R12C: a new Smads protein phosphorylation pathway is involved in regulation of TGF beta signaling activity; SQSTM1 interaction as a scaffold protein and other proteins mediated by Smads play regulatory role in the signaling pathway; in addition to the screening interaction Smad4 and NLK, suggesting that Wnt/TGF beta signaling pathway may play a new interactive contact.
In addition, this paper makes a preliminary study on the Smad3 complex, the tandem affinity purification method for purification of Smad3 complex were identified in isolated liver related cells. First constructed retroviral vector of target gene, then transfected into packaging cell clones were screened, the results to be identified.
In summary, the important group in the TGF signaling pathway were studied interacting proteins by using Y2H technology. Through the interaction of other methods of verification, analysis and preliminary experimental study on the function of bioinformatics, presumably some novel interacting proteins screened may play an important role in the regulation of TGF beta signaling pathway. Provide clues for exploring the pathway to play the new mechanism function.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R363

【共引文獻】

相關(guān)期刊論文 前4條

1 譚世明;李桂源;;TSC-22基因的研究進展[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2011年07期

2 范媛;詹tg;劉杰;;口腔扁平苔蘚基因表達譜中差異表達基因初步探討[J];華西口腔醫(yī)學(xué)雜志;2007年04期

3 劉青蘭;許雋永;曾娟;范媛;;轉(zhuǎn)化生長因子β刺激基因22在口腔扁平苔蘚組織中的表達及意義[J];口腔醫(yī)學(xué);2010年07期

4 曾娟;劉青蘭;蔡葉;范媛;;玉屏風(fēng)口服液對口腔扁平苔蘚差異表達基因干預(yù)的初步探討[J];口腔醫(yī)學(xué);2010年08期

本文編號：1442684