核蛋白NDP52与肿瘤坏死因子受体相关因子TRAF6相互作用及临床意义的研究

发布时间：2018-03-28 16:34

本文选题：蛋白质相互作用　切入点：NDP52　出处：《安徽医科大学》2011年硕士论文

【摘要】：核因子kappa B(nuclear factor kappa B, NF-KB)是近年来颇受关注的一组重要因子,作为可诱导并普遍存在的转录因子,对众多基因发挥中心性转录调节作用,从而在免疫、炎症、细胞的生存、增殖、分化和凋亡、肿瘤的形成等方面起到广泛而重要的作用。NDP52是ND10(核蛋白)组成成员之一,该蛋白在肝组织中表达量最高,能够与其它分子相互作用最终引起细胞生命效应,与病毒的感染,炎症的形成,肿瘤的发生发展有明确的相关性。研究发现,该蛋白可能与肿瘤坏死因子受体相关因子6(TRAF6)共同参与NF-KB信号通路的调节,但调控机制尚未清除。为进一步阐明NDP52和TRAF6在NF-KB信号通路中的调控机制,及其与肝癌发生发展的关系,我们以酵母双杂交筛选成人肝脏cDNA文库得到的未知相互作用对NDP52与TRAF6为研究对象,通过一系列实验验证了两者的相互作用,并为阐明NF-κB信号通路的分子机制及肝癌发生机制提供了新的线索。前期实验以TRAF6为诱饵质粒,采用酵母双杂交技术筛选成人肝脏cDNA文库得到相互作用对NDP52与TRAF6。我们通过外源免疫共沉淀实验,GST-Pull down实验和和荧光共定位实验等确认NDP52和TRAF6间存在生理性相互作用。转录活性实验发现,过表达NDP52时,对TRAF6介导的NF-κB信号起着负调控作用,随着NDP52量的增加,这种负调控逐渐增强。除了TRAF6以外,NDP52与TRAF6家族的其它成员TRAF1,TRAF2,TRAF3和TRAF5也存在相互作用,其相互作用也可以影响NF-κB的转录活性。通过Western blot实验检测了NDP52在不同肝癌细胞和其他癌细胞中的表达。细胞免疫荧光实验证实了NDP52蛋白在恶性黑色素瘤A375,肝癌细胞(HCC cell)(7721)中表达,细胞质和细胞核中都有分布,主要在细胞核内分布。肝细胞癌免疫组化实验结果看,NDP52蛋白在肝癌组织的细胞核表达量有有上升的趋势,而在正常对照的肝组织中表达量非常少。同时我们采用酶联免疫吸附实验实验检测了肝细胞肝癌病人血清中NDP52蛋白含量的多少。ELISA实验结果显示,在肝细胞肝癌病人血清中NDP52蛋白含量高于对照组正常人血清中NDP52的含量。NDP52与TRAF6存在生理性相互作用,在探知NDP52和TRAF6的相互作用是否影响NF-κB信号通路时,我们发现过表达NDP52时,对TRAF6介导的NF-κB信号起着负调控作用,随着NDP52量的增加,这种负调控逐渐增强。除了TRAF6以外,NDP52与TRAF6家族的其它成员TRAF1,TRAF2,TRAF3和TRAF5也存在相互作用,其相互作用也可以影响NF-κB的转录活性。确定NDP52与TRAF6在体内存在相互作用后,我们推测NDP52与TRAF6应该有相同的亚细胞定位,免疫荧光实验验证了我们的推测,NDP52与TRAF6相互作用的生理功能是发生在细胞质中。通过Western blot实验检测了NDP52在不同肝癌细胞和其他癌细胞中的表达,从选择的几种肝癌细胞的裂解液来看,在几种肝癌细胞中均有NDP52的表达;而在其他几种非肝癌的癌细胞中NDP52也有表达,说明NDP52并非肝癌细胞特异性表达蛋白。细胞免疫荧光实验证实了NDP52蛋白在恶性黑色素瘤(A375),肝癌细胞(7721)中表达,细胞质和细胞核中都有分布,但主要是在细胞核。从肝细胞癌免疫组化实验结果看,NDP52蛋白在I级、II级、III级肝癌组织的细胞核表达量有有上升的趋势,与肿瘤的分化程度似乎相关联,而在正常对照的肝组织中表达量非常少。同时我们采用酶联免疫吸附实验检测了20例肝细胞肝癌病人血清中NDP52蛋白含量,ELISA实验结果显示,在肝细胞肝癌病人血清中NDP52蛋白含量高于对照组正常人血清中NDP52的含量。本研究发现NDP52对NF-κB信号通路具有抑制作用,而且这种负调控作用很可能是在TRAF6的水平来实现的,这为阐明NF-κB信号通路的分子机制提供新的线索。明确了NDP52与TRAF6相互作用的亚细胞定位,同时发现NDP52与肝细胞肝癌相关,填补了NDP52与何种疾病相关这一空缺,让我们重新认识了NDP52新的功能角色,为进一步的NDP52功能研究奠定了坚实的基础。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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