本文選題：蛋白質(zhì)相互作用　切入點：NDP52　出處：《安徽醫(yī)科大學(xué)》2011年碩士論文

【摘要】：核因子kappa B(nuclear factor kappa B, NF-KB)是近年來頗受關(guān)注的一組重要因子,作為可誘導(dǎo)并普遍存在的轉(zhuǎn)錄因子,對眾多基因發(fā)揮中心性轉(zhuǎn)錄調(diào)節(jié)作用,從而在免疫、炎癥、細(xì)胞的生存、增殖、分化和凋亡、腫瘤的形成等方面起到廣泛而重要的作用。NDP52是ND10(核蛋白)組成成員之一,該蛋白在肝組織中表達量最高,能夠與其它分子相互作用最終引起細(xì)胞生命效應(yīng),與病毒的感染,炎癥的形成,腫瘤的發(fā)生發(fā)展有明確的相關(guān)性。研究發(fā)現(xiàn),該蛋白可能與腫瘤壞死因子受體相關(guān)因子6(TRAF6)共同參與NF-KB信號通路的調(diào)節(jié),但調(diào)控機制尚未清除。為進一步闡明NDP52和TRAF6在NF-KB信號通路中的調(diào)控機制,及其與肝癌發(fā)生發(fā)展的關(guān)系,我們以酵母雙雜交篩選成人肝臟cDNA文庫得到的未知相互作用對NDP52與TRAF6為研究對象,通過一系列實驗驗證了兩者的相互作用,并為闡明NF-κB信號通路的分子機制及肝癌發(fā)生機制提供了新的線索。前期實驗以TRAF6為誘餌質(zhì)粒,采用酵母雙雜交技術(shù)篩選成人肝臟cDNA文庫得到相互作用對NDP52與TRAF6。我們通過外源免疫共沉淀實驗,GST-Pull down實驗和和熒光共定位實驗等確認(rèn)NDP52和TRAF6間存在生理性相互作用。轉(zhuǎn)錄活性實驗發(fā)現(xiàn),過表達NDP52時,對TRAF6介導(dǎo)的NF-κB信號起著負(fù)調(diào)控作用,隨著NDP52量的增加,這種負(fù)調(diào)控逐漸增強。除了TRAF6以外,NDP52與TRAF6家族的其它成員TRAF1,TRAF2,TRAF3和TRAF5也存在相互作用,其相互作用也可以影響NF-κB的轉(zhuǎn)錄活性。通過Western blot實驗檢測了NDP52在不同肝癌細(xì)胞和其他癌細(xì)胞中的表達。細(xì)胞免疫熒光實驗證實了NDP52蛋白在惡性黑色素瘤A375,肝癌細(xì)胞(HCC cell)(7721)中表達,細(xì)胞質(zhì)和細(xì)胞核中都有分布,主要在細(xì)胞核內(nèi)分布。肝細(xì)胞癌免疫組化實驗結(jié)果看,NDP52蛋白在肝癌組織的細(xì)胞核表達量有有上升的趨勢,而在正常對照的肝組織中表達量非常少。同時我們采用酶聯(lián)免疫吸附實驗實驗檢測了肝細(xì)胞肝癌病人血清中NDP52蛋白含量的多少。ELISA實驗結(jié)果顯示,在肝細(xì)胞肝癌病人血清中NDP52蛋白含量高于對照組正常人血清中NDP52的含量。NDP52與TRAF6存在生理性相互作用,在探知NDP52和TRAF6的相互作用是否影響NF-κB信號通路時,我們發(fā)現(xiàn)過表達NDP52時,對TRAF6介導(dǎo)的NF-κB信號起著負(fù)調(diào)控作用,隨著NDP52量的增加,這種負(fù)調(diào)控逐漸增強。除了TRAF6以外,NDP52與TRAF6家族的其它成員TRAF1,TRAF2,TRAF3和TRAF5也存在相互作用,其相互作用也可以影響NF-κB的轉(zhuǎn)錄活性。確定NDP52與TRAF6在體內(nèi)存在相互作用后,我們推測NDP52與TRAF6應(yīng)該有相同的亞細(xì)胞定位,免疫熒光實驗驗證了我們的推測,NDP52與TRAF6相互作用的生理功能是發(fā)生在細(xì)胞質(zhì)中。通過Western blot實驗檢測了NDP52在不同肝癌細(xì)胞和其他癌細(xì)胞中的表達,從選擇的幾種肝癌細(xì)胞的裂解液來看,在幾種肝癌細(xì)胞中均有NDP52的表達;而在其他幾種非肝癌的癌細(xì)胞中NDP52也有表達,說明NDP52并非肝癌細(xì)胞特異性表達蛋白。細(xì)胞免疫熒光實驗證實了NDP52蛋白在惡性黑色素瘤(A375),肝癌細(xì)胞(7721)中表達,細(xì)胞質(zhì)和細(xì)胞核中都有分布,但主要是在細(xì)胞核。從肝細(xì)胞癌免疫組化實驗結(jié)果看,NDP52蛋白在I級、II級、III級肝癌組織的細(xì)胞核表達量有有上升的趨勢,與腫瘤的分化程度似乎相關(guān)聯(lián),而在正常對照的肝組織中表達量非常少。同時我們采用酶聯(lián)免疫吸附實驗檢測了20例肝細(xì)胞肝癌病人血清中NDP52蛋白含量,ELISA實驗結(jié)果顯示,在肝細(xì)胞肝癌病人血清中NDP52蛋白含量高于對照組正常人血清中NDP52的含量。 本研究發(fā)現(xiàn)NDP52對NF-κB信號通路具有抑制作用,而且這種負(fù)調(diào)控作用很可能是在TRAF6的水平來實現(xiàn)的,這為闡明NF-κB信號通路的分子機制提供新的線索。明確了NDP52與TRAF6相互作用的亞細(xì)胞定位,同時發(fā)現(xiàn)NDP52與肝細(xì)胞肝癌相關(guān),填補了NDP52與何種疾病相關(guān)這一空缺,讓我們重新認(rèn)識了NDP52新的功能角色,為進一步的NDP52功能研究奠定了堅實的基礎(chǔ)。
[Abstract]:Nuclear factor kappa B (nuclear factor kappa B, NF-KB) is a group of important factors of popular concern in recent years, as a transcription factor can be induced and universal, a number of genes play a central role in transcriptional regulation, resulting in immune, inflammation, cell survival, proliferation, differentiation and apoptosis, tumor formation to play the role of.NDP52 is extensive and important ND10 (nuclear protein) composed of a member of the protein in the highest expression in liver tissue, can interact with other molecules eventually lead to cell life effects, and the virus infection causes inflammation, there is a clear correlation between the occurrence and development of tumors. The study found that the protein and tumor necrosis factor receptor associated factor 6 (TRAF6) involved in regulating the NF-KB signaling pathway, but regulatory mechanisms are not yet clear. To further elucidate the mechanism of NDP52 regulation and TRAF6 in NF-KB signaling, and With the occurrence and development of HCC, we use yeast two hybrid screening adult liver cDNA Library of unknown interaction between NDP52 and TRAF6 as the research object, through a series of experiments to verify the interaction between the two, and provides new clues for elucidating the molecular mechanism of NF- B signaling pathway and liver cancer early experimental mechanism. Using TRAF6 as a bait plasmid, by screening a human liver cDNA library by yeast two hybrid technique by interaction between NDP52 and TRAF6. by exogenous CO immunoprecipitation, GST-Pull down experiments and fluorescence co localization of the physical interaction experiments confirmed that NDP52 and TRAF6. The experiment found that the transcriptional activity, the overexpression of NDP52 plays a negative. Regulation of NF- kappa B signaling mediated by TRAF6, with the increase of NDP52 content, the negative regulation gradually. In addition to TRAF6, TRAF NDP52 and other members of the TRAF6 family 1, TRAF2, TRAF3 and TRAF5 are interaction, the interaction can also affect the transcriptional activity of NF- K B. Through Western blot analysis to detect the expression of NDP52 in different hepatoma cells and other cancer cells. Immunofluorescence experiments confirmed that NDP52 protein in malignant melanoma cells (HCC A375, cell) (7721) expression, all has the distribution in the cytoplasm and the nucleus, mainly distributed in the nucleus. Hepatocellular carcinoma immunohistochemistry results showed that NDP52 protein in HCC cell nucleus expression has a rising trend, and in normal liver tissue expression is small. At the same time we used enzyme ELISA assay of NDP52 protein in hepatocellular carcinoma patient serum.ELISA the number of experimental results show that in serum in patients with hepatocellular carcinoma NDP52 protein content higher than that of the control group of normal human serum NDP52 The physical interaction of.NDP52 and TRAF6 content, whether in the interaction of NDP52 and TRAF6 to ascertain the effect of NF- B signaling pathway, we found that overexpression of NDP52, plays a negative regulatory role of NF- kappa B signaling mediated by TRAF6, with the increase of NDP52 content, the negative regulation gradually. In addition to TRAF6 NDP52, TRAF6 and other members of the family TRAF1, TRAF2, TRAF3 and TRAF5 are interaction, the interaction can also affect the transcriptional activity of NF- K B. NDP52 and TRAF6 determine the in vivo interaction, we speculate that NDP52 and TRAF6 should have the same subcellular localization, immunofluorescence experiments validate our presumably, the physiological function of NDP52 and TRAF6 interaction occurs in the cytoplasm. The Western blot analysis to detect the expression of NDP52 in different hepatoma cells and other cancer cells, from the selected cell lysis Liquid, both the expression of NDP52 in HCC cell; and in several other non liver cancer cells also expressed NDP52, the expression of NDP52 is not the liver cell specific protein. Immunofluorescence experiments confirmed that NDP52 protein in malignant melanoma (A375), liver cancer cells (7721) expression. There are distributed in the cytoplasm and nucleus, but mainly in the nucleus. From hepatocellular carcinoma immunohistochemistry results showed that II NDP52 protein in the I level, III level, liver tissue expression of nucleus has a rising trend, and the degree of tumor differentiation seems to be associated, and in normal liver tissue the expression is small. At the same time we used enzyme-linked immunosorbent assay in serum of 20 cases of hepatocellular carcinoma in patients with NDP52 protein detection, ELISA results showed that in serum in patients with hepatocellular carcinoma NDP52 protein content is higher than normal control group The content of NDP52 in human serum.
The study found that NDP52 can inhibit NF- B signaling pathway, and this negative regulation is likely to be achieved at the level of TRAF6, which provides new clues to elucidate the molecular mechanism of NF- B signaling pathway. The subcellular localization of NDP52 interaction with TRAF6, and found that NDP52 and liver cells liver cancer related, NDP52 related diseases and to fill the vacancy, let us re understand the functional role of new NDP52, which laid a solid foundation for the further study on the function of NDP52.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R363

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