Survivin蛋白和LMP1在鼻咽癌组织中的表达关系

目的：探讨凋亡抑制基因Survivin蛋白和EB病毒潜伏膜蛋白（LMP1）在鼻咽癌组织中的表达情况以及它们之间的关系.方法：应用免疫组织化学技术S-P法对68例鼻咽癌组织标本进行凋亡抑制基因Survivin蛋白与EB-LMP1的表达检测.结果：鼻咽癌组织Survivin蛋白产物的阳性率为82.4%（56/68）,LMP1阳性率为67.6%（46/68）;56例Sur-vivin蛋白表达的阳性鼻咽癌标本中,有42例LMP1阳性,共同阳性率75.0%（42/56）,12例Survivin蛋白表达的阴性鼻咽癌标本中,9例LMP1阴性,共同阴性率66.7%（9/12）,两者之间高度相关（P〈0.01）.结论：鼻咽癌组织中凋亡抑制基因Survivin蛋白的表达与LMP1的表达密切相关.     

鼻咽癌活检组织中EB病毒膜抗原和EB病毒核酸的分析

应用抗EB病毒膜抗原(EBV/MA)的单克隆抗体检测51例病理确诊的鼻咽癌(NPC)活检标本的冰冻切片,其中49例有EBV/MA的表达。经细胞形态学分析首次发现鼻咽癌癌细胞,增生上皮细胞以及正常鼻咽部上皮细胞都有EBV/MA。以Southern杂交法检查20例鼻咽癌活检组织中EB病毒核酸(EBV/DNA),结果证实鼻咽癌组织中不仅有EB病毒核酸的重复序列W片段,还有与细胞转化有关的K片段的序列存在。进一步表明EB病毒在鼻咽癌的发生中并非“过客”,很可能是其病原因素之一。     

Detection and analysis of anti-latent membrane protein 2A antibodies in the sera of patients with Epstein-Barr virus associated malignancies

Background Epstein-Barr virus (EBV) associated malignancies with a Type Ⅱ latency gene expression pattern, such as Hodgkin‘s disease, and nasopharyngeal carcinoma (NPC) , frequently express the EBV antigen latent membrane protein 2A (LMP2A). We expected to establish a highly expressing LMP2A yeast cell strain and get the high quality LMP2A protein, which was used for detection, analysis and characterization of its antibodies in various patients‘ sera of EBV associated malignancies. Methods The plasmid pPICZαA-LMP2A containing the full length of LMP2A cDNA was constructed and transformed to Pichia pastoris GS115 to express LMP2A protein. After fermentation and purification, the LMP2A protein was used as an antigen to detect anti-LMP2A antibodies (Abs) in the sera of patients with EBVassociated malignancies in enzyme linked immunosorbent assay (ELISA) or Western-blot. Results LMP2A was expressed successfully with an expected molecular weight of approximately 54 kD and Abs to LMP2A were strikingly specific to NPC. Two-thirds or more sera from NPC patients were positive for antiLMP2A immunoglobulin G (IgG) Abs. The antibodies were absent from the sera of other EBV-associated diseases except a small fraction of the gastric carcinoma. Comparing anti-viral capsid Ags (VCA) IgA and LMP2A IgA titers in the sera from 76 NPC patients, only 55% were positive for anti-LMP2A IgA Abs while 70% were positive for anti-VCA IgA. However, we found that 3 sera negative for VCA IgA were positive for LMP2A IgA. Conclusion The results suggested the potential significance of LMP2A specific Abs for the diagnosis of EBVassociated malignancies, especially NPC.     

P53和P21蛋白在鼻咽癌中的表达及其临床意义

目的：探讨P53和P21蛋白在鼻咽癌（NPC）、癌旁组织和鼻咽慢性炎症中表达的差异，及其与肿瘤生物学行为的关系。材料与方法：首程放疗前的NPC病人100例，癌旁组织和鼻咽慢性炎症对照组各20例，用SP法进行P53和P21蛋白免疫组化染色，P53阳性细胞的标准为：癌巢内肿瘤细胞核有棕色沉着的细胞；P21阳性细胞的标准为：癌巢内肿瘤细胞浆有棕色沉着的细胞。结果：在鼻咽癌、癌旁组织和鼻咽慢性炎症组织中，P53蛋白的阳性率分别为：78．0％、35．0％和0．0％，P＝0．001；P21蛋白的阳性率分别为：83．0％、75．0％和65．0％，P＝0．170。在鼻咽癌原发灶早期（T1＋2）和晚期（T3＋4），颈结阴性和阳性及临床早期（Ⅰ Ⅱ）和临床晚期（Ⅲ Ⅳ）各组中，P53蛋白和P21蛋白的阳性率均无差异（P＞0．05）。结论：P53基因的突变与鼻咽癌的发生有一定的关系。P53蛋白可能是鼻咽癌癌病变的指标；P21的突变可能与鼻咽癌的发生无明显的关系，P53蛋白和P21蛋白的阳性率与鼻咽癌原发灶的早晚，颈结转移，和临床分期无关。     

NPC相关抗原的血清学分析

目的:对重组克隆表达抗原血清学分析(SEREX)法筛选出的鼻咽癌(NPC)系列肿瘤抗原进行特异性检测,判断新检测出的NPC抗原的相应抗体在NPC病人血清及正常人血清中的阳性率,为NPC抗原对NPC的免疫诊断、免疫治疗积累基础资料。方法:运用噬菌体转染-蛋白印迹、免疫组织化学技术,检测3种NPC肿瘤抗原(GX-NPC-1,GX-NPC-2,GX-NPC-4)在20个NPC病人及15个正常人血清中的阳性表达率。结果:3种NPC肿瘤抗原在NPC病人血清中的阳性表达率分别为55%(11/20)、55%(11/20)、50%(10/20);正常人各为33.3%(5/15)、26.7%(4/15)、6.7%(1/15),经统计学分析,前两种肿瘤抗原在NPC及正常人血清中的表达差异无统计学意义,而GX-NPC-4在两种血清中的表达差异有统计学意义(P<0.01)。结论:3种NPC肿瘤抗原中GX-NPC-4抗原在NPC患者的血清中的表达率明显增高,可能为NPC特异性抗原,值得进一步研究。     

Primary nasopharyngeal non-Hodgkin lymphoma and its relationship with Epstein-Barr virus infection

Objectives To investigate the immunophenotypes of primary nasopharyngeal non-Hodgkin lymphoma(NPL) and their relationship to Epstein-Barr virus (EBV) infection.Methods The clinical data and biopsies of 73 patients with NPL were collected in Guangzhou. In situ hybridization was performed to detect the EBV-encoded small non-polyadenylated nuclear RNAs(EBERs) on biopsy slides. Immunohistochemistry was used to classify the immunophenotypes of NPL and detect EBV antigen expression.Results Forty-four (60. 27%) of the 73 NPLs were of B cell lineage (CD79α^ /CD3^-/CD56^-)while the 29 others (39.73%) were of non-B cell lineage. Seventy-three NPLs could be classified into 3 major immunophenotypes: B cell (CD79α^ /CD3^-/CD56 ^-, 44 cases), peripheral T cell (CD79α^-/CD3^ /CD56^-, 22) and NK/T cell (CD79α^-/CD3^ /CD56^ , 7). The percentages of EBV infection differed among the 3 major immunophenotypes ( B cell : 11.36%, 5/44 ; peripheral T cell: 81.82%,18/22; NK/T cell: 100%, 7/7). Both CD56-positive and CD56-negative immunophenotypes could further be divided into 4 subtypes: CD8^-/CD4^-, CD8^ /CD4^- , CD8^-/CD4^ and CD8^ /CD4^ All the CD8^-/CD4^- NPLs with CD56-positivity (7) or CD56-negativity (2) were infected with EBV. The neoplastic cells of a nasopharyngeal Burkitt‘ s lymphoma expressed EBV nuclear antigen 1 (EBNA1)and EBV RNA (EBERs) only. In the other 29 EBV-infected NPLs, most of the lymphoma cells harboring EBV also expressed EBNA1 and EBERs; 21 of the 29 NPLs had a considerable number of neoplastic cells expressing latent membrane protein 1 ( LMP1 ) (21/29, 72.41% ) and 23 of 29 NPLs expressed latent membrane protein 2A (LMP2A) (23/29, 79. 31%). A few lymphoma cells in 17( 17/29, 58. 62% ), 23 (23/29, 79.31% ) and 22 NPLs (22/29, 75.86% ) expressed Zta ( Barn HI Z transactivator), viral capsid antigen (VCA) and membrane antigen (MA), respectively.Conclusions The prevalence ratio of the 3 immunophenotypes, namely, B cell, peripheral T cell and NK/T cell lymphoma, is about 6:3: 1. However, the EBV infection ratio is reversed, 1:8: 10. All the NK/T cell (CD56^ ) and peripheral immature T cell (CD3^ /CD8^-/CD4^-) NPLs were EBV-infected.Except for one Burkitt‘ s lymphoma, the EBV harbored in both B cell and non-B cell NPLs was mainly latent infection, type It, expressing EBNA1, LMP1 and LMP2A. However, the EBV found in a few lymphoma cells could become replicative, expressing lytic proteins.     

鼻咽癌组织中EBER-1表达及其临床病理学意义

目的：探讨爱泼斯坦-巴尔病毒(EBV)与鼻咽癌发生、发展的关系及其临床病理学意义。方法：应用免疫组化技术检测46例鼻咽癌、28例鼻咽癌癌旁组织、36例慢性鼻咽炎石蜡切片组织中爱泼斯坦-巴尔病毒受体(EBER-1)的表达。结果：鼻咽癌组织中EBER-1表达率为74．O％，且在转移灶中不丢失，癌旁组织、慢性鼻咽炎组织中EBER-1表达率分别为32．1％、16．7％。结论：检测EBER-1的表达对鼻咽癌诊断与鉴别诊断具有重要价值。慢性鼻咽炎组织、癌旁组织中：EBER-1阳性者提示为高危癌前病变，应密切随访监测，可能有利于早期发现鼻咽癌。     

鼻咽癌中EB病毒潜伏膜蛋白1启动子的突变

 【目的】 检测EB病毒潜伏膜蛋白1 (LMP1) 远端启动子L1-TR在鼻咽癌(NPC)组织中的突变情况并探讨突变对启动子活性的影响。【方法】 用聚合酶链反应(PCR)方法分别扩增出B95.8细胞和新鲜鼻咽癌组织细胞中EB病毒(EBV)的L1-TR片段,并测序比较其核苷酸序列的差异。构建含原型和突变型L1-TR启动子的荧光素酶报告质粒并分别转染HaCat细胞、B95.8细胞和C666-1细胞,比较两种启动子的活性。【结果】 和B95.8原型比较,鼻咽癌组织中的EB病毒L1-TR启动子区域有多处缺失。插入和点突变,且启动子活性明显降低(P < 0.05)。【结论】 鼻咽癌组织中LMP1启动子 L1-TR的活性与B95.8原型相比明显降低,这在EB病毒的免疫逃避机制中可能有一定的意义。     

EB病毒感染与宿主细胞恶性转化的关系

EB病毒(EBV)是一种与多种肿瘤发生密切相关的DNA肿瘤病毒。EBV基因表达产物有潜伏膜蛋白(LMP1、LMP2A、LMP2B),6种核抗原(EBNA1、EBNA2、EBNA3A、EBNA3B、EBNA3C、EBNA-LP),以及EBER1、EBER2和TP等。EBV通过影响和调控宿主细胞基因的表达,干扰细胞信号转导系统,使宿主细胞产生细胞因子表达失衡。EBV能上调IRF-4、IL-6、Bcl-6、Bcl-2、A20等细胞生长因子、抗凋亡因子,还能抑制p53和PTPPK等肿瘤抑制基因,促使宿主细胞转化为肿瘤细胞。     

鼻咽癌患者血清中EB病毒四种特异性IgG抗体的测定

用间接免疫荧光法检测了２４例鼻咽癌患者与１１９例正常人血清中ＥＢ病毒４种特异性ＩｇＧ抗体，结果表明，正常人血清中ＥＢ病毒衣壳抗原（ＷＣＡ）的抗体效价为１：１６０以下，而鼻咽癌患者血清抗体效价为ｌ：１６０～１：５１２０，抗体阳性率均为１００％；正常人血清中ＥＢ病毒核抗原（ＥＢＮＡ）的抗体效价为１：８０以下，而鼻咽患者血清抗体价为１：８０～１：２５６０，阳性率均为１００％；正常人血清中ＥＢ病毒早期抗原（ＥＡ）的抗体效价为ｌ：１０，阳性率只有１３．３％，而鼻咽癌患者血清抗体效价为ｌ：２０～１：１２８０，阳性率为１００％；正常人血清中ＥＢ病毒抗原（ＭＡ）的抗体效价为１：１０～１：４０，阳性率为７１％，而鼻咽癌患者血清抗体效价为１：１０～１：３２０，阳性率为１００％．以上４种抗体中．ＶＣＡ、ＥＢＮＡ和ＥＡ抗体的效价和分布在正常人与鼻咽癌患者之间有明显差异（Ｐ＜姜勇男，男，３１岁，讲师．延吉市局子街２１号（１３３０００）０．０ｌ），ＥＡ抗体的阳性率也比正常人明显升高（Ｐ＜０．０ｌ），ＭＡ抗体的效价和阳性率与正常人相比无显著差异（Ｐ＞０．０５）。     

重组腺相关病毒介导RNA干扰抑制EB病毒潜伏膜蛋白1对鼻咽癌细胞影响的动物试验

目的 探讨EB病毒(EBV)潜伏膜蛋白1(LMP-1)对于鼻咽癌细胞在体内增殖及转移能力的影响.方法 设计针对EBV-LMP-1的特异性发夹状RNA(shRNA)干扰序列,构建2种重组腺相关病毒(rAAV)载体:(增强型绿色荧光蛋白,EGFP)rAAV-EGFP和rAAV-shRNA-LMP-1,以不同滴度rAAv-EGFP转染鼻咽癌C666-1细胞确定最佳转染复数(MOI),rAAV-shRNA-LMP-1按MOI转染C666-1,RT-PCR鉴定抑制效率,将体外转染RNA干扰后的C666-1细胞注入裸鼠肝脏包膜下制作鼻咽癌异位肝种植的肝肺转移模型,观察肝脏成瘤及肝内、肺转移情况,分析LMP-1基因沉默在动物水平对鼻咽癌细胞成瘤及转移能力的影响.结果 rAAV-EGFP以5×104v.g(virus genome,病毒基因组数)/细胞转染C666-1细胞,转染效率大于95%,RT-PCR鉴定rAAV-shRNA-LMP-1以5×104v.g/细胞转染C666-1后目的 基因抑制效率大于90%,动物试验结果显示rAAV-shRNA-LMP-1组肝脏成瘤体积与对照组rAAV-EGFP无显著差异,但显著减少了种植肿瘤的肝内及肺转移率.结论 通过rAAV介导RNA干扰能有效抑制LMP-1基因表达,可在动物水平抑制鼻咽癌细胞转移,但对鼻咽癌细胞生长无显著影响.     

Recombinant Vaccinia Virus is an Effective and Non—perturbing Vector for Human Dendritic Cells Transfected with Epstein—Barr Virus Latent Membrane Protein 2A

Objective To study the effects of dendritic cells(DC) transfected with recombinant vaccinia virus encoding Epstein-Barr virus(EBV) latent membrane protein 2A(LMP2A) gene,and to provide evidence for further investigation on the therapeutic uaccines against EBV-associated malignancies.Methods Mature DC were transfected with EVB-LMP2A recombinant vaccinia virus(rVV-LMP2A).Before and after the transfection,the expression of surface antigens on mature DC including CD1a,CD83,CD40,CD80,HLA-DR was measured by fluorescence activated cell sorter(FACS) and the function of DC to stimulate allogeneic T cells proliferation was measured by mixed leukocyte reactions(MLR).Results LMP2A protein was highly expressed (66.1%) in DC after the transfection of rVV-LMP2A.No significant changes in the primary surface antigens expression and in the MLR were detected during the transfection.Transfected DC still had strong potential in stimulating the proliferation of allogeneic T cells.Conclusion Recombinant vaccinia virus was an effective and non-perturbing vector to mediate the transfection of LMP2A into DC.The functions of mature DC were not affected significantly by the transfection of Vac-LMP2A.This study could provide evidence for the further immunotherapy of EBV-associated malignancies,e.g.nasopharyngeal carcinoma(NPC).     

年轻人霍奇金淋巴瘤亚型特点及肿瘤细胞EB病毒和Bcl-2蛋白的表达及其意义

目的:研究EB病毒(EBV)与Bcl-2蛋白在年轻人霍奇金淋巴瘤肿瘤细胞中的表达,阐明二者之间的关系.方法:复查48例16～36岁霍奇金淋巴瘤患者的临床和病理资料,应用免疫组织化学方法检测肿瘤细胞中CD30、CD15、CD45、LMP-1和Bcl-2蛋白的表达并进行组织学分型;采用组织芯片的原位杂交方法检测EBV的表达...     

Epstein-Barr virus infection in nasopharyngeal lymphoid hyperplasia

Ｏｂｊｅｃｔｉｖｅ　ＴｏｄｅｔｅｃｔｗｈｅｔｈｅｒＥｐｓｔｅｉｎＢａｒｒｖｉｒｕｓ（ＥＢＶ）ｈａｒｂｏｒｓｉｎｎａｓｏｐｈａｒｙｎｇｅａｌｌｙｍｐｈｏｉｄｈｙｐｅｒｐｌａｓｉａ（ＮＰＬＨ）ｗｈｉｃｈｉｓｆｒｅｑｕｅｎｔｌｙｔｏｂｅｓｅｅｎｉｎＧｕａｎｇｚｈｏｕ,ａｈｉｇｈｉｎｃｉｄｅｎｃｅａｒｅａｏｆｎａｓｏｐｈａｒｙｎｇｅａｌｃａｒｃｉｎｏｍａ（ＮＰＣ）,ａｎｄｔｏｅｘｐｌｏｒｅｔｈｅｒｅｌａｔｉｏｎｂｅｔｗｅｅｎＮＰＬＨａｎｄｄｅｖｅｌｏｐｍｅｎｔｏｆＮＰＣＭｅｔｈｏｄｓ　Ｔｗｅｎｔｙ…     

EB病毒壳抗原在大肠杆菌中的表达及纯化

本文报告对EB病毒壳抗原(VCA)带有抗原决定簇的蛋白质在大肠杆菌中的表达及其纯化。以纯化后的蛋白为抗原检测人血清中VCA/IgA抗体,鼻咽癌病人及某些急性EB病毒感染人群呈抗体阳性.而对照组呈阴性。这一结果提示VCA蛋白在鼻咽癌的普查中有应用价值。     

Epstein-Barr virus encoded latent membrane protein 1 induces TRAF1 expression to promote anti-apoptosis activity via NF-κB signaling pathway in nasopharyngeal carcinoma

Objectives To identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1(LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-KB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).Methods A stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.Results LMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMPI. Moreover, LMPl-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the KB site KB1 or KB5 was disrupted,whereas mutation of κB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.Conclusion LMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-κB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.     

融合基因GM-CSF-BZLF1重组腺病毒表达载体的构建

目的构建粒细胞-巨噬细胞集落刺激因子(GM-CSF)和EB病毒即刻早期基因(BZLF1)融合基因的重组腺病毒表达载体。方法采用逆转录-聚合酶链反应分别获得GM-CSF和BZLF1编码序列的cDNA,应用剪接式重叠延伸(SOE)技术将两段基因通过多肽接头(Gly4Ser)3的DNA序列进行连接,构建融合基因GM-CSF-BZLF1。将融合基因GM-CSF-BZLF1定向亚克隆至pAdTrack-CMV质粒,在原核细胞E.coliBJ5183中完成穿梭质粒与骨架质粒pAdEasy-1的同源重组,构建融合基因GM-CSF-BZLF1真核表达载体pAd-GM-CSF-BZLF1。将真核表达载体pAd-GM-CSF-BZLF1转染293细胞,获得复制缺陷型重组腺病毒vAd-GM-CSF-BZLF1。RT-PCR鉴定感染重组腺病毒的293细胞中GM-CSF-BZLF1基因的表达。结果 GM-CSF-BZLF1基因插入重组腺病毒表达载体的预期位置,且插入序列完全正确;感染重组腺病毒vAd-GM-CSF-BZLF1的293细胞中检测到融合基因GM-CSF-BZLF1的转录表达。结论成功地构建了融合基因GM-CSF-BZLF1重组腺病毒表达载体,为进一步探讨GM-CSF-BZLF1的功能提供了理论基础和实验依据。     

鼻咽癌血清中EB病毒核抗原-1和核抗原-2抗体的研究

用抗补体免疫荧光法检测鼻咽癌患者和对照人群中EB病毒核抗原-1(EBNA-1)和EB病毒核抗原-2(EBNA-2)抗体的水平。鼻咽癌组中两种抗体滴度都升高,以EBNA-1抗体上升明显;且EBNA-1抗体与总EBNA抗体水平的变化趋势相一致。鼻咽增生性病变组中EBNA-2抗体略占优势,该组中EBNA-2抗体水平升高可能与潜伏状态的EB病毒激活或再感染的早期阶段有关。     

EB病毒相关胃癌及鼻咽癌组织病毒主要潜伏期基因启动子甲基化状态分析

目的研究分析青岛地区EB病毒（EBV）相关胃癌及鼻咽癌组织中EBV主要潜伏期编码基因启动子甲基化状态。方法采用甲基化特异性PCR（MSP）及亚硫酸氢盐-基因组测序法（BGS）,分析32例EBV相关胃癌及20例EBV阳性鼻咽癌标本中编码EBV核抗原EBNAs基因的启动子Cp、Wp、Qp以及LMP1和LMP2A编码基因启动子的甲基化状态。结果 EBV相关胃癌及鼻咽癌肿瘤标本中EBV编码基因启动子的甲基化状态与肿瘤细胞的潜伏感染类型一致：Cp和Wp呈高甲基化状态,而Qp未发生甲基化;2种EBV阳性肿瘤标本中LMP1和LMP2A编码基因启动子甲基化状态有所不同,两者在EBV相关胃癌组织甲基化程度明显高于其在EBV阳性的鼻咽癌（χ^2=19.15、11.01,P〈0.01）。结论甲基化是调控EBV主要潜伏期基因启动子活动的重要机制之一。     

应用RNA干扰抑制EB病毒潜伏膜蛋白-1表达对鼻咽癌细胞生长的影响

目的探讨能否通过siRNA人工诱导RNA干扰,在EB病毒阳性的鼻咽癌细胞株C611中,选择性抑制潜伏膜蛋白1(LMP1)的表达,并了解LMP1抑制对鼻咽癌细胞生长的影响。方法利用脂质体转染的方法,将人工合成的双链siRNA导入鼻咽癌细胞,通过半定量RT-PCR检测LMP1 mRNA水平的变化。并采用MTT法和流式细胞仪检测LMP1表达抑制后,鼻咽癌细胞增殖和细胞周期的变化。结果C611细胞转染siRNA可使LMP1 mRNA水平下降90%以上。LMP1基因抑制后,C611细胞增殖速度下降33%;流式细胞检测显示,C611细胞周期在G0-G1期受阻。结论本研究证明,全新的人工诱导RNA干扰技术,能够有效抑制LMP1基因的表达,并降低鼻咽癌细胞的增殖能力,为鼻咽癌研究和抗肿瘤基因治疗提供了新的思路。