【作者】 刘洁琼；

【导师】 贺智敏；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 背景与目的:EB病毒（Epstein-Barr virus,EBV）是一种在人类普遍流行的γ-疱疹病毒,与许多人类肿瘤如伯基特淋巴瘤、霍奇金淋巴瘤、胃癌、鼻咽癌等的发生发展密切相关。其中EBV潜伏膜蛋白1（LMP1）被公认为病毒基因组编码的唯一具有促细胞转化作用的瘤蛋白,在鼻咽癌发生过程中可能发挥重要作用。LMP1的羧基末端（aa187～386）是LMP1致瘤作用的主要部位,包括3个活化区域（carboxy terminal activation region,CTAR）:即CTAR1（194～231aa）、CTAR2（TRADD结合区351～386aa）与CTAR3（275～330aa）。研究表明,LMP1能持续活化NF-kB,AP-1,JAKs/STATs并通过这些分子激活下游信号传导通路而产生致瘤效应。由于蛋白质磷酸化是信号传导通路中最广泛最重要的翻译后修饰,而且这些通路中的许多信号分子及其磷酸化状态还远远没有阐明。因此,为了全面了解、验证LMP1介导的信号通路,阐明CTAR1、CTAR2与CTAR3在LMP1促鼻咽上皮细胞转化成瘤中所涉及的信号网络分子,本研究拟采用磷酸化蛋白质组学方法分离和鉴定LMP1转化的鼻咽上皮细胞的差异磷酸化蛋白,为EB病毒在鼻咽癌发生发展中的作用及机制提供进一步证据。方法:采用脂质体转染法将野生型LMP1、2个突变型LMP1（TRADD位点突变和aa232～351缺失）的逆病毒载体pLNSX-LMP1^WT、pLNSX-LMP1^TRADD与pLNSX-LMP1^Δ232-351（以pLNSX为对照）分别导入PA317包装细胞,G418筛选2周后扩大培养,自细胞培养上清获得后续实验所需的感染性逆病毒RV-pLNSX、RV-LMP1^WT、RV-LMP1^TRADD与RV-LMP1^Δ232-351。然后,用浓缩的病毒分别感染人鼻咽上皮细胞NP69,汇合克隆培养,获得NP69-pLNSX、NP69-LMP1^WT、NP69-LMP1^TRADD与NP69-LMP1^Δ232-3514种转化细胞系。观察和检测以上转化细胞的形态、生长曲线、平板克隆、细胞周期等。同时,采用固相pH梯度双向凝胶电泳技术结合Western Blot方法分析NP69-pLNSX、NP69-LMP1^WT、NP69-LMP1^TRADD与NP69-LMP1^Δ232-351细胞系磷酸化蛋白质表达的差异,即在感染24小时后抽提细胞总蛋白,2-DE分别分离NP69-pLNSX、NP69-LMP1^WT、NP69-LMP1^TRADD与NP69-LMP1^Δ232-351细胞的总蛋白质,每组平行进行两块胶电泳,其中一块作为制备胶,另一块作为分析胶。制备胶考染显色,得到了分辨率较高、重复性较好的NP69-pLNSX、NP69-LMP1^WT、NP69-LMP1^TRADD与NP69-LMP1^Δ232-351细胞总蛋白质2-D胶电泳图谱;分析胶中的蛋白质转移至硝酸纤维素膜后,与抗酪氨酸磷酸化抗体孵育,进行WesternBlot分析,获得差异表达酪氨酸磷酸化蛋白质的反应图谱;将制备胶的电泳图谱和Western Blot的反应图谱进行比对分析,在制备胶上找到相应的差异酪氨酸磷酸化蛋白质点。利用MALDI-TOF-MS对差异酪氨酸磷酸化蛋白质进行鉴定,部分差异蛋白质应用免疫沉淀方法进行验证。结果:（1）NP69-LMP1^WT细胞在培养过程中,逐渐由多角形、鹅卵石样典型上皮形态演变成细胞间接触减少的长梭状纤维细胞样形态,而NP69-LMP1^TRADD与NP69-LMP1^Δ232-351细胞呈近乎多角形,比较接近NP69-pLNSX细胞形态;NP69-LMP1^WT细胞的生长速度、S期细胞所占比例比NP69-LMP1^TRADD与NP69-LMP1^Δ232-351及载体对照组NP69-pLNSX细胞明显增加;（2）在NP69-pLNSX与NP69-LMP1^WT细胞比较组鉴定了12种差异表达的磷酸化蛋白质（上调有HSP27、波形蛋白等,下调有膜联蛋白A1、GTP结合蛋白等）,在NP69-LMP1^WT与NP69-LMP1^TRADD细胞比较组鉴定了11种差异表达的磷酸化蛋白质,在NP69-LMP1^WT与NP69-LMP1^Δ232-351细胞比较鉴定了10种差异表达的磷酸化蛋白质。其中大部分是与细胞结构、信号传导和转录翻译相关的蛋白;（3）在这些差异蛋白质中,有10种磷酸化蛋白质体现了LMP1羧基端各功能区的特异性,如HSP27在NP69-LMP1^WT与NP69-LMP1^TRADD细胞表达增加（与NP69-pLNSX细胞比较）,在NP69-LMP1^Δ232-351细胞却不增加等。（4）免疫沉淀验证了4种蛋白在转化细胞蛋白磷酸化表达水平的差异,结果与蛋白质组学结果一致结论:（1）LMP1可能通过上调HSP27、波形蛋白、角蛋白等,下调膜联蛋白A1、GTP结合蛋白,分子伴侣TCP1等多种蛋白的磷酸化促进NP69细胞转化。（2）CTAR2可能介导LMP1对波形蛋白,ER-60,HSP70等蛋白磷酸化的上调和膜联蛋白A1,磷脂酰乙醇胺结合蛋白,蛋白酶体等蛋白磷酸化的下调而产生致瘤作用;（3）CTAR3可能介导LMP1对ER-60,角蛋白8,HSP27等蛋白磷酸化的上调和磷脂酰乙醇胺结合蛋白,蛋白酶体,膜联蛋白A2等蛋白磷酸化的下调而产生致瘤作用。更多还原

【Abstract】 Background and Objective Epstein-Barr virus（EBV）is a prototype gamma herpes virus that infects the majority of the population worldwide and has been implicated in the pathogenesis of several human malignancies including Burkitt’s and Hodgkin’s lymphomas,gastric carcinoma and nasopharyngeal carcinoma（NPC）.EBV-encoded latent membrane protein 1（LMP1）is considered a classic oncoprotein because of its ability to transform rodent fibroblast cell lines and drive the immortalization of primary human B-lymphocytes in vitro.So it was the first EBV latent gene found to be able to transform cell lines and alter the phenotype of cells due to its oncogenic potential.The C-terminal regions of LMP1 is the main signal activated position which can be subdivided into three essential C-terminal-activating regions（CTARs）:C-terminal activation regions 1,2 and 3（CTAR 1,CTAR2 and CTAR3）.Previous studies have shown that many of the oncogenic effects of LMP1 can be explained by its ability to constitutively activate nuclear factor kappa B （NF-kB）,activator protein-1（AP-1）,JAKs/ STATs.Due to protein phosphorylation is the most widespread and capital post-translational modification,and many signaling molecules and downstream target proteins mediated by the oncoprotein LMP1 in epithelial cells, particularly NPC cells,are largely unknown.In order to globally understand and confirm the signal pathways mediated by LMP1,to explain the molecular net involved in LMP1 CTAR1,CTAR2 and CTAR3-mediated transformation and tumorigenesis of nasopharyngeal epithelial cells,this study is to plan to separate and identify the differential phosporylated protein associated with LMP1 transforming nasopharyngeal epithelial cells by phosphoproteomics approach,and to provide further evidence for the role of EBV on the development of NPC.Method pLNSX（as controlled vector）,pLNSX-LMP1^WT、pLNSX-LMP1^TRADDand pLNSX-LMP1^Δ232-351were transfected into the ecotropic retrovirus packaging cell line PA317 with lipofectamine, respectively.The transfected PA317 cells were selected with G418 sulfate, two weeks later,the resistant cells were collected and expanded as the virus-producing cell lines(RV-pLNSX,RV-LMP1^WT、RV-LMP1^TRADDand RV-LMP1^Δ232-351)to use in subsequent experiment.Then,nasopharyngeal epithelium cells NP69 were infected by the concentrated retrovirus respectively.The stable transfected cell lines NP69-pLNSX, NP69-LMP1^WT,NP69-LMP1^TRADDand NP69-LMP1^Δ232-351were observd and detected cellular morphology,cellular growth curve,colony formation and cell cycle.Meanwhile,2-DE coupled with Western Blot method were used to analysis of the differentially expressed tyrosine-phosphorylated proteins among NP69-pLNSX,NP69-LMP1^WT, NP69-LMP1^TRADD,NP69-LMP1^Δ232-351cell lines.After infected 24 hours, The total proteins of the four groups were separated by 2-DE,respectively. Each patch was separated in parallel on analytical as well as preparative 2-D gels.The preparative 2-D gels were stained by Goomassie Brilliant Blue.Well-resolved and reproducible 2-D preparative patterns of NP69-pLNSX,NP69-LMP1^WT,NP69-LMP1^TRADD,NP69-LMP1^Δ232-351 cell lines were acquired.The analytical gels were electroblotted onto a nitrocellulose membrane and incubated with anti-phosphotyrosine antibody.After Western Blot analysis,2-D maps of differentially expressed tyrosine-phosphorylated proteins were acquired.After comparing the preparative maps with Western Blot maps,differentially tyrosine-phosphorylated proteins were found in corresponding preparative gels.Differentially tyrosine-phosphorylated proteins between the four cell lines were identified using MALDI-TOF-MS analysis and database searching.The differential expression levels of the partial proteins were determined by Western blot.Result（1）The morphous of NP69-LMP1^WTcells gradually developed from a typical epithelial polygon,cobblestone to an elongated and fibroblastoid shape with a marked reduction in cell-cell contact.But NP69-LMP1^TRADDand NP69-LMP1^Δ232-351exhibited approximately polygonal morphous and grew in a more compact pattern with tighter cell-cell contact.The growth velocity and the proportion of S stage of NP69-LMP1^WTcells obviously increased than NP69-LMP1^TRADD、 NP69-LMP1^Δ232-351cells and NP69-pLNSX cells;（2）12 differentially tyrosine-phosphorylated proteins between NP69-pLNSX and NP69-LMP1^WTcell lines（up-regulation proteins as heat shock protein 27, vimentin ea al,down-regulation proteins as annexin A1,chaperonin cotaining TCP1 et al）;11 differentially tyrosine-phosphorylated proteins between NP69-LMP1^WTand NP69-LMP1^TRADDcell lines;10 differentially tyrosine-phosphorylated proteins between NP69-LMP1^TRADDand NP69-LMP1^Δ232-351cell lines Most of them were characterized as cellular structure proteins,signal transduction and transcription and translation associated proteins.（3）Among these identified phosphoproteins,there were 10 proteins personificated the specificness of the C-terminal-activating regions of LMP1.For example, the expression of HSP27 in NP69-LMP1^WTand NP69-LMP1^TRADDcell lines was much more than NP69-pLNSX ones.However,there was no expression in NP69-LMP1^Δ232-351cell lines.This indicated the CTAR3 having the main contribution in this process.（4）In the transformed cells, 2 proteins were confirmed with immunoprecipitation on the level of protein expression.Both results were coincident with the results of the proteomics.Conclusions（1）LMP1 promotes NP69 cell transforming with the up-regulation of heat shock protein 27,vimentin and CK7 and the down-regulation of annexin A1,chaperonin cotaining TCP1 and GTP binding protein et al.（2）The CTAR2 domain of LMP1 was pivotal participating in the above effects with the up-regulation of the expression phosphorylation of vimentin,ER-60,HSP70 annexin A1 and down-regulation of phosphorylation of phosphotidylethanolamin binding protein,proteasome et al to generate tumorigennesis contribution;（3）The CTAR3 domain of LMP1 was pivotal participating in the above effects with the up-regulation of the expression of phosphorylation of ER-60, cytoskeletal 8,HSP70 and down-regulation of phosphorylation of phosphotidylethanolamin binding protein,proteasome,annexin A2 et al to generate tumorigennesis contribution.更多还原