CD21非依赖性EB病毒对人胃印戒细胞癌细胞系的感染

目的　探讨CD2 1非依赖性EB病毒 (EBV)对人胃印戒细胞癌细胞系 (HSC 39)的感染作用。方法　用Akata和P3HR 1EBV毒株感染HSC 39,有限稀释法对感染细胞进行克隆。结果　两种EBV毒株感染细胞中均可检测到EBV编码的小RNA(EBER)的表达 ,两种EBV毒株感染的亲代细胞及大多数细胞克隆表达EBV核抗原 (EBNA1) ,但不表达EBNA2、潜伏期膜蛋白 (LMP1)和LMP2A。表现为潜伏Ⅰ型感染。未感染的HSC 39细胞及P3HR 1感染的细胞克隆CD2 1表达阴性 ,而AkataEBV感染的部分细胞克隆CD2 1mRNA阳性。结论　EBV可能通过不依赖CD2 1受体的途径感染HSC 39,印戒细胞癌细胞系可用作EBV感染的靶细胞。     

EBV潜伏基因产物在恶性淋巴瘤组织中的表达及意义

目的：探讨EBV潜伏基因产物在恶性淋巴瘤组织中的表达及意义。方法：用免疫组化方法对565例NHL、64例HL人体标本进行LMP－1、EBNA2对比检测并选择101例NHL进行PCR检测。结果：EBV－PCR检出率（19．8％）高于LMP－1（14．9％）,PCR阴性病例LMP－1全部为阴性,EBNA2在全部病例均为阴性,在NHL,LMP－1阳性细胞主要是免疫母细胞样细胞、R－S样细胞和R－S细胞,LMP－1阳性的R－S样细胞我数表达活化分子CD30。肠道原发恶性淋巴瘤EBV检出率较高（23．1％）。T淋巴瘤EBV检出率（23．8％）高于B淋瘤（10．2％）。结论：EBV潜伏基因产物表达情况能够反映出宿主细胞的分化程度和（或）宿主的免疫监视作用。EVB在R－S样细胞形成可能起作用。EBV感染与肠道恶性淋巴瘤的发病有关。     

EB病毒潜伏膜蛋白2的研究回顾

Epstein Barr病毒 (EBV)属于疱疹病毒科γ亚科 ,在人类中广泛传播。大多数EBV的初次感染是在患者幼儿时期 ,而且没有明显的临床症状 ,终身携带病毒。EBV与越来越多的人类肿瘤相关 ,包括由于免疫抑制引起的免疫增生性淋巴瘤、Burkitt淋巴瘤、NPC、HD和多种T细胞淋巴瘤等疾病。EBV潜伏感染时表达 8种蛋白 (6种核蛋白EBNA1、EBNA2、EBNA3A、EBNA3B、EBNA3C、EBNALP和 2种膜蛋白LMP1和LMP2 )。在这些与转化相关的病毒蛋白中 ,EBNA1、LMP1、LMP2是在NPC肿瘤标本和EBV相关肿瘤中可以检测到的蛋白。1　LMP2的功能198…     

EBV潜伏膜蛋白LMP2A的研究进展

EB病毒（Epstein-Barr virus,EBV）与多种人类恶性肿瘤如Burkitt淋巴瘤、鼻咽癌（na-sopharyngeal carcinoma,NPC）、何杰金氏病（Hodgkin＇s disease,HD）等的发生有关,肿瘤组织中EBV主要以潜伏感染的形式存在。在EBV潜伏感染细胞中,可表达的EBV基因有10多种,包括EBV核抗原（EB nuclear antigen,EBNA）1,2,3A,3B,3C和LP;潜伏膜蛋白（latent membrane protein,LMP）1,2A和2B;EBV编码的小RNA（EBER）1和2;以及BamHⅠ-A向右开放读码框（BARF0）的转录产物。NPC和其他EBV相关恶性肿瘤中可持续检测到LMP2A的转录物,提示LMP2A在体内病毒持续感染和EBV相关疾病中可能有重要作用。     

EB病毒感染对鼻咽癌细胞生长和凋亡的影响

目的:探讨EB病毒(EBV)感染对鼻咽癌细胞系生长和细胞凋亡的影响。方法:用EBV直接感染人鼻咽癌细胞系CNE1;用免疫组化(LSAB)法检测EBV-LMP1和bcl-2蛋白的表达;用MTT法测定鼻咽癌细胞系的生长能力;用流式细胞术和TUNEL法检测癌细胞凋亡。结果:感染EBV的鼻咽癌细胞系(E-CNE1)的EBV-LMP1表达阳性,生长能力较未感染EBV的CNE1明显增强(P<0.01),2种鼻咽癌细胞系均无凋亡发生,而均仅有2%~3%的细胞表达bcl-2蛋白。结论:EBV感染和LMP1表达可促进鼻咽癌细胞生长,但对鼻咽癌细胞的bcl-2表达和细胞凋亡无影响。     

EB病毒感染对鼻咽癌细胞生长和凋亡的影响

目的:探讨EB病毒(EBV)感染对鼻咽癌细胞系生长和细胞凋亡的影响。方法:用EBV直接感染人鼻咽癌细胞系CNE1;用免疫组化(LSAB)法检测EBV-LMP1和bcl-2蛋白的表达;用MTT法测定鼻咽癌细胞系的生长能力;用流式细胞术和TUNEL法检测癌细胞凋亡。结果:感染EBV的鼻咽癌细胞系(E-CNE1)的EBV-LMP1表达阳性,生长能力较未感染EBV的CNE1明显增强(P＜0.01),2种鼻咽癌细胞系均无凋亡发生,而均仅有2%~3%的细胞表达bcl-2蛋白。结论:EBV感染和LMP1表达可促进鼻咽癌细胞生长,但对鼻咽癌细胞的bcl-2表达和细胞凋亡无影响。     

EB病毒LMP1在鼻咽癌细胞系中通过NF-κB、AP-1促进IL-8分泌

目的：探讨EB病毒LMP1分子致瘤机制，在已证实鼻咽癌细胞系中LMP1有效激活NF－κB 或AP－1的基础上，对LMP1是否通过NF－κB 或AP－1促进IL－8分泌进行探讨。方法：以稳定表达LMP1及其3种突变体，空白载体的鼻咽癌细胞系[HNE2-LMP1,NHE2-MLP1(1-185),HNE2-LMP1(1-231),HNE2-LMP1Δ187-351和HNF3－pSG5]及antisense-LMP1处理的HNF2－LMP1鼻咽癌的细胞系为材料，将IL－8报道质粒瞬时导入这些细胞系中，通过测定luciferase值以反映LMP1是否促进IL－8转录；将mut AP-1/IL-8-luc或IκB α（S32A／S36A）表达质粒导入HNE2－LMP1细胞系中，比较其IL－8报道活性，以确定LMP1是否通过AP－1或NF－κB 诱导IL－8转录；利用ELISA方法测定HNE2－LMP1，HNE2－pSG5,anti-sese-LMP1处理的HNE2－LMP1鼻咽癌细胞系中的IL－8浓度，进一步从蛋白水平上确定LMP1是否促进IL－8分泌。结果：与HNE2－pSG5相比，在HNE2－LMP1，HNE2－LMP1Δ187－351和HNE2－LMP1（1－231）细胞系中IL－8报道活性分别升高了原来水平的11．5，8．6和3．4倍，而HNE2－LMP1（1－185）对IL－8报道活性不影响。在HNE2－LMP1细胞系中IL－8蛋白水平提高了17．4倍，而antisense-LMP1则使HNE2－LMP1细胞的IL－8报道活性及蛋白水平分别下降到原来水平的18．3％和9．2％，导入mutAP-1/IL-8-luc或IκBα（S32A／S36A）的HNE2－LMP1细胞中IL－8报道活性分别下降到原有水平39％和26％，结论：鼻咽癌细胞系中LMP1可能通过NF－κAP－1促进IL－8表达。     

RNA激发的DC体外诱导EBV特异性CTL的研究

本研究采用酶切等方法将质粒pSVTP1中LMP2a (latentmembraneprotein 2a)的编码基因克隆至pGEM 3Zf+,再经体外转录系统转录获得EBV LMP2aRNA ,将以此RNA激发的DC所诱导生成的CTL作为效应细胞分别与含EBV基因之一EBNA1、EBNA2、EBNA3c、LMP2a的重组病毒感染的正常人成纤维细胞混合后 ,采用LDH释放法检测细胞毒活性。结果显示 ,经体外转录的LMP2aRNA激发的DC所诱导的淋巴细胞对表达LMP2a抗原的成纤维细胞产生特异性的细胞毒活性 ,证实了这种RNA激发的DC能有效地诱导EBV特异性CTL的生成 ,为EBV相关肿瘤的免疫治疗提供了新的实验依据。     

潜伏期膜蛋白1与疾病关系的研究进展

EB病毒(EBV)是一种普遍存在的致癌病毒,是高度相关的淋巴和上皮肿瘤的来源和发展,包括伯基特淋巴瘤和鼻咽癌(NPC)等,EBV基因几乎可以在所有细胞中。EBV感染通常与少数潜伏病毒功能的蛋白质表达,包括潜伏膜蛋白LMP1和LMP2A等和巴尔核抗原1(EBNA1)。LMP1是肿瘤坏死因子受体超家族的成员,被认为是EBV的主要致瘤蛋白。在EB病毒中通过2个C末端结构域编码基因蛋白LMP1信号来驱动细胞生长,存活和转化。LMP1蛋白目前是惟一已被证实的EB病毒的癌基因,LMP1的表达参与了肿瘤的发生与发展,是目前癌症方面研究的重点。     

腺病毒载体介导EB病毒潜伏期膜蛋白2A基因转染树突状细胞对其功能的影响

目的 探讨腺病毒载体介导EB病毒（EBV）潜伏期膜蛋白2A（LMP2A）基因转染树突状细胞（DC）对其功能的影响。方法 通过同源重组法构建带有EBV－LMP2A基因的重组腺病毒Ad5-LMP2A，用不同感染滴度（MOI）的Ad5-LMP2A转染成熟的DC，用流式细胞术（FACS）检测DC的LPM 2A蛋白表达细胞百分率及用台盼蓝染色计数DC死亡百分率，选择最佳MOI；用最佳MOI的Ad5-LMP2A转染成熟的DC，FACS检测转染前后DC表面分子CD1a、CD83、CD40、CD80及HLA－DR的变化；并用^3H-TdR掺入法检测转染前后DC刺激同种淋巴细胞增殖能力及荧光定量PCR检测表达IL－12 P40 mRNA等功能的改变。结果 MOI 200为Ad5-LMP2A转染DC的最佳滴度，此时约80％的DC表达LMP2S蛋白及92％以上细胞为活细胞。Ad5-LMP2A转染成熟DC前后对细胞表面共刺激分子及特征性表面标志无影响；转染后的DC仍具有较强的刺激同种异体淋巴细胞增殖能力和表达IL－12 P40 mRNA的功能。结论 腺病毒载体能高效介导EBV LMP2A基因在DC中表达，Ad5-LMP2A转染成熟DC对其功能无明显影响，便于进一步用于EBV相关肿瘤的免疫治疗。     

CD21-independent Infection of Epstein-Barr Virus in Human Signet Ring Gastric Carcinoma Cell Line

Epstein Barr virus ( EBV), a ubiquitous human her pesvirus, is the etiologic agent of infectious mononucleo sis, and is closely associated with several human malig nancies including Burkitt′s lymphoma (BL), undifferenti ated nasopharyngeal carcinoma (NPC) and opportunisticlymphoma in immunocompromised hosts. In recent years,there have been increasing evidences of association of EBVwith additional malignancies, such as gastric carcinoma.EBV has been found in tumor cells of m…     

A naturally derived gastric cancer cell line shows latency I Epstein-Barr virus infection closely resembling EBV-associated gastric cancer

In a process seeking out a good model cell line for Epstein-Barr virus (EBV)-associated gastric cancer, we found that one previously established gastric adenocarcinoma cell line is infected with type 1 EBV. This SNU-719 cell line from a Korean patient expressed cytokeratin without CD19 or CD21 expression. In SNU-719, EBNA1 and LMP2A were expressed, while LMP1 and EBNA2 were not. None of the tested lytic EBV proteins were detected in this cell line unless stimulated with phorbol ester. EBV infection was also shown in the original carcinoma tissue of SNU-719 cell line. Our results support the possibility of a CD21-independent EBV infection of gastric epithelial cells in vivo. As the latent EBV gene expression pattern of SNU-719 closely resembles that of the EBV-associated gastric cancer, this naturally derived cell line may serve as a valuable model system to clarify the precise role of EBV in gastric carcinogenesis.     

The vIL-10 gene of the Epstein-Barr virus (EBV) is conserved in a stable manner except for a few point mutations in various EBV isolates

A gene of the Epstein-Barr virus (EBV), BamHI-C fragment rightward reading frame 1 (BCRF1), codes viral interleukin-10 (vIL-10), which is a close homolog to human IL-10. EBV strain variations are known at EBV latent membrane protein 1 (LMP1), and the distinct forms of LMP1 have been identified. In order to further elucidate the variations of EBV strains, the BCRF1 (vIL-10) gene was analyzed using PCR-direct sequencing in African Burkitt’s lymphoma (BL) cell lines Raji, P3HR-1, EB1 and Daudi, Japanese BL cell line Akata, lymphoblastoid cell line OB and 22 wild EBV isolates from eight gastric carcinoma tissues and 14 throat washes. We found only five variations of the vIL-10 gene in them with one silent mutation and three non-silent mutations. Raji had no mutation to the prototype gene of B95-8. EB1 and P3HR-1 had non-silent mutations in the sequences leading to the arginine/serine and threonine/proline interchanges at residues 4 and 166, respectively. The silent mutation was detected at valine 102 in Daudi and also in the Japanese cell lines Akata, OB and 20 (90.9%) of the wild EBV isolates. The type of variations in the vIL-10 gene had a common relationship with those in the LMP1 gene. All of the variants of valine 102 had China1-type LMP1 sequences except for Daudi with Med-type LMP1 and other minorities with B95-8 type LMP1. The conservativeness of vIL-10 with a few variations suggests the indispensability of the vIL-10 gene in EBV and that the variations of the vIL-10 gene may depend upon the geographical prevalence of the EBV strains. This is the first report regarding the variations of the vIL-10 gene in cell lines and other wild isolates.     

Heterogeneity of Epstein-Barr virus. II. Induction of early antigens (EA) by complementation.

Cells of Epstein-Barr virus (EBV)-negative human B lymphoma lines BJA and Ramos were converted into EBV genome carriers by virus isolates from P3HR-1 and B 95-8 cells (Fresen and zur Hausen, 1976). Cloning of P3HR-1 virus-converted BJA cells resulted in clones with two different Epstein-Barr nuclear antigen (EBNA) patterns: a faint granular EBNA staining and clones with a brilliant EBNA expression (Fresen et al., 1977). The latter always segregated EBNA-negative cells from which one EBNA-negative subclone (B1-28) was isolated. Induction of early antigens (EA) was studied by infecting parental lines (BJA and Ramos), converted lines (BJA-HR1K, BJA-B 95-8, Ramos-HR1K, Ramos-B 95-8), the BJA-HR1K clones A5 (faint granular EBNA expression) and B1-19 (brilliant EBNA expression), the EBNA-negative subclone B1-28, and Raji cells with EBV from P3HR-1 and B 95-8 cells, respectively. The following results were obtained: (1) EA induction by P3HR-1 virus is enhanced on the average 14-fold in EBV genome-harboring cells when compared to genome-negative lines. (2) B 95-8 virus induces EA only in P3HR-1 virus-converted cells and to a small extent also in Raji cells. A significant EA induction occurs in the A5 clone of BJA-HR1K, whereas the brilliantly EBNA-expressing B1-19 clone is not induced. B 95-8 virus-converted cells cannot be induced by B 95-8 virus. (3) EA induction following infection of EBV genome-carrying cells is directly proportional to the dilution of the infecting virus. In EBV genome-free cells, EA induction is reduced by the square of the dilution factor. These results imply that resident genomes complement superinfecting genomes in EA induction by EBV and that two different populations of genomes (present in P3HR-1 virus isolates) are required for EA induction following infection of B lymphoblasts.     

Growth transformation of primary epithelial cells with a NPC-derived Epstein-Barr virus strain

The Epstein-Barr virus (EBV) is associated with two major human epithelial malignancies, where it is likely to play a role in the malignant phenotype: undifferentiated nasopharyngeal carcinoma (100% of cases) and gastric carcinomas (about 10% of cases). We and others have obtained growth transformation of monkey kidney primary epithelial cells by transfection of viral DNA, especially with the BARF1 gene of EBV (Wei et al., 1997). We now report that the same type of primary epithelial cells can be growth-transformed using EBV particles derived from a nasopharyngeal carcinoma tumor line. Not only can these EBV-infected cells grow over 100 passages, escaping senescence, in contrast to their noninfected counterparts, but they can also survive and proliferate at very low cell density. Several subclones were characterized in terms of viral gene expression. All these clones gave a similar pattern, with detection of EBNA1 and BARF1 proteins but absence of LMP1. CD21, which is the main EBV receptor on B lymphocytes, was not expressed on parental monkey kidney epithelial cells nor on EBV-infected cell clones. This model of epithelial cell transformation will be useful for a better investigation of EBV functions critical for oncogenesis of epithelial cells.     

Contrasting effects of hydroxyurea on cell growth and reduction in Epstein-Barr virus genomes in EBV-infected epithelioid cell lines vs Burkitt's lymphoma cell lines

Eliminating Epstein-Barr virus (EBV) genomes from infected cells is an intriguing theoretical strategy in therapy for EBV-associated malignant diseases. Respective patterns were characterized for hydroxyurea (HU)-promoted loss of EBV genomes from EBV-infected epithelioid cell lines derived from the noncancerous portion of gastric carcinoma tissues and Burkitt's lymphoma (BL) cell lines. Epithelioid cell lines GT38 and PN were less sensitivity to HU than BL cell lines Akata, Raji, and Daudi in terms of cell growth inhibition and cell cycle arrest. On passage in medium with 50 microM HU, the fraction of EBV nuclear antigen (EBNA)-positive cells was reduced substantially in the BL cell lines, but only slightly in the epithelioid cell lines. EBV DNA was reduced in Akata, Raji, and Daudi cells upon passage in 50 microM HU by 95%, 70%, and 50%, respectively, but by only 10% in GT38 cells, in which EBV DNA reduction was enhanced at increased concentrations of HU. This indicates that EBV genome is more easily lost from BL cell lines than from epithelioid cell lines upon culturing in HU. These findings support the view that the elimination of EBV could be therapeutically effective in EBV-associated BL by HU.     

Effect of transforming growth factor-beta1 on the cell growth and Epstein-Barr virus reactivation in EBV-infected epithelial cell lines.

Transforming growth factor (TGF)-beta1 is a multifunctional cytokine that plays important roles in regulating cell growth and differentiation in many biological systems. In this study, we found that gastric tissue-derived Epstein-Barr virus (EBV)-infected epithelial cell lines GT38 and GT39 had resistance to TGF-beta1-mediated growth inhibition and apoptosis compared to a TGF-beta1-susceptible gastric carcinoma cell line HSC-39. However, TGF-beta1 partially induced EBV reactivation in GT38 and GT39 cells, as shown by the induction of EBV immediate-early BZLF1 RNA and its protein product ZEBRA and early antigen-D. The expressions of TGF-beta receptor I and II were detected in GT38 and GT39 cells by Northern and Western blot analyses. Both cell lines spontaneously produced the TGF-beta1, which was sufficient for inhibiting cell growth of HSC-39 cells. Taken together, these data suggest that TGF-beta1 may be a key factor for EBV reactivation and selective growth of EBV-infected epithelial cells in vivo.     

Distinct Patterns of Mitogen-Activated Protein Kinase Phosphorylation and Epstein–Barr Virus Gene Expression in Burkitt's Lymphoma Cell Lines versus B Lymphoblastoid Cell Lines

In order to understand mechanistic relationships between signaling pathways regulating mitogen-activated protein kinase (MAPK) phosphorylation and Epstein–Barr virus (EBV) reactivation, we compared MAPK phosphorylation, and EBV reactivation and latency in Burkitt's lymphoma cell lines (BLCLs) versus B lymphoblastoid cell lines (LCLs). EBV was reactivated in the BLCLs Akata and Raji, and in a LCL OB-R33 cells after cross-linking surface immunoglobulin (sIg) with anti-Ig. After stimulation with anti-Ig, MAPK phosphorylation was strongly induced in all BLCLs and in a few LCLs, but not in other LCLs. MAPK was constitutively phosphorylated in most LCLs but not in BLCLs. Expression of EBNA2 and LMP1, and LMP2A was analyzed with both immunoblotting and RT-PCR. EBNA2 and LMP1 were expressed in most LCLs and in some BLCLs. LMP2A was expressed in all BLCLs and LCLs except Namalwa cells. To test the hypothesis that LMP1 induces constitutive MAPK phosphorylation, the LMP1 expression vector was transfected into Akata cells. MAPK phosphorylation was not induced in such transfected cells. Our results indicate that BLCLs and LCLs respectively have distinct MAPK phosphorylation patterns, and that induction of MAPK phosphorylation correlates with EBV reactivation in a few cell lines but not in most of the tested cell lines.