中草药抑制EB病毒壳抗原在体外细胞中表达的实验研究

目的:探讨中草药对EB病毒壳抗原在体外细胞中表达的抑制作用.方法:采用间接免疫酶法检测广西常见的300余种中草药对B95-8细胞壳抗原表达的抑制作用.结果:发现其中20种中草药有抑制EB病毒抗原表达的作用.结论:抑制EB病毒壳抗原在体外细胞中表达的中草药有望在鼻咽癌预防方面起一定作用.     

黄芪对Epstein—Barr病毒抗原表达的抑制作用

目的：探讨黄芪对EB病毒（EBV）抗原表达的抑制作用。方法：采用间接免疫酶法研究黄芪提取液对Raji细胞和B95-8细胞EBV抗原表达的抑制作用。结果：无细胞毒性浓度的黄芪提取液对巴豆油，正丁酸联合激发的EB病毒早期抗原（EA），壳抗原（VCA）表达均有明显抑制作用，抑制率随药物浓度增加而提高。结论：黄芪抑制EBV抗原表达效果好，有望在鼻咽癌的预防方面有一定作用。     

黄芪对Epstein-Barr病毒抗原表达的抑制作用

目的 :探讨黄芪对EB病毒 (EBV)抗原表达的抑制作用。方法 :采用间接免疫酶法研究黄芪提取液对Raji细胞和B95 8细胞EBV抗原表达的抑制作用。结果 :无细胞毒性浓度的黄芪提取液对巴豆油、正丁酸联合激发的EB病毒早期抗原 (EA)、壳抗原 (VCA)表达均有明显抑制作用 ,抑制率随药物浓度增加而提高。结论 :黄芪抑制EBV抗原表达效果好 ,有望在鼻咽癌的预防方面起一定作用。     

富硒米提取液对EB病毒转化脐血B淋巴细胞及EBV早期抗原表达的影响

背景与目的:硒是人体必需的微量元素,具有抗氧化、抗衰老和抑制肿瘤细胞增殖的作用,为进一步探讨硒在肿瘤防治中的作用,我们培育出富含有机硒的大米,现研究富硒米对EB病毒激发脐血B淋巴细胞转化及对Raji细胞EB病毒早期抗原表达的影响。方法:①用富硒米及普通米提取液以1∶4、1∶8的浓度分别加到制备好的EB病毒液中,加入脐血单个核细胞进行淋巴母细胞转化培养实验,计算细胞转化抑制率;②Raji细胞经正丁酸及巴豆油联合诱导的同时加富硒米提取液共同培养,以间接荧光法,经伊文氏蓝复染观察计算EBV-EA抗原阳性率和抑制率。结果:含硒0.11μg/ml(1∶8稀释)的富硒米提取液显著抑制EB病毒对脐血B淋巴细胞的转化,抑制率为83.4%,明显高于普通米提取液的抑制率61.3%(P<0.05)。富硒米提取液对Raji细胞EB病毒早期抗原(earlyantigen,EA)表达有明显抑制作用,0.016、0.078、0.388μg/ml硒浓度组对EA抗原抑制率分别为2.85%、12.88%、20.75%。结论:富硒米提取液对EB病毒转化脐血B淋巴细胞和对Raji细胞EB病毒早期抗原均有明显的抑制作用。     

亚硒酸钠对Epstein-Barr病毒抗原表达影响的研究

本文报告亚硒酸钠在试管对两株携带Epstein—Barr病毒基因组的类淋巴母细胞株Raji和B_(95-8)细胞株的活力及EB病毒抗原表达的影响。实验表明0.01—0.1μg/mlNa_2SeO_3对两株细胞的活力几乎无影响,1—10μg/ml则对细胞显示不同程度的毒性,Se对B_(95-8)的细胞株的生长抑制较对Raji细胞株的抑制为弱。无细胞毒性的0.01—0.1μg/mlNa_2SeO_3对正丁酸巴豆油协同诱导的Raji细胞EA抗原和B_(95-8)细胞的VCA抗原表达呈现显著抑制作用。8小时硒溶液预处理后对EA抗原激发的抑制以及硒对B_(95-8)细胞的自发VCA抗原的抑制试验说明抑制是作用于靶细胞,而硒对B_(95-8)细胞诱发的VCA抗原的抑制明显强于自发VCA抗原,这提示Se对抗原激发物有一定的影响。 大量研究报告,硒具有抗病毒诱发的肿瘤和化学致癌作用,由于鼻咽癌和EB病毒密切相关,对硒抑制EB病毒基因表达与细胞转化关系的深入研究将有可能为了解EB病毒与肿瘤的关系提供重要线索。     

树突状细胞经EB病毒膜抗原gp340刺激后诱导抗肿瘤免疫应答

目的：探索利用树突状细胞（dendritic cells，DC）制备针对EB病毒感染相关性肿瘤的疫苗，以解决EB病毒相关性肿瘤在机体内的免疫逃逸。方法：来源于正常人骨髓的CD34+造血干/祖细胞，体外以重组hGMCSF，hTNFα，hIL4，hIL3诱导培养2周，扩增出功能成熟的DC，经EB病毒表面膜糖蛋白gp340刺激，探讨DC诱导T淋巴细胞介导免疫应答的能力，观察负载抗原gp340 DC诱导出的抗原特异性CTL对EB病毒相关性肿瘤细胞的杀伤作用。结果：从人骨髓细胞中诱导扩增出的DC具有良好的免疫刺激活性；EB病毒表面膜糖蛋白gp340可有效负载DC，DC加工处理抗原后激发T淋巴细胞增殖反应的能力进一步增强；在EB病毒相关性肿瘤细胞的杀伤实验中，只有负载了抗原gp340的DC，才能刺激特异性CTL杀伤EB病毒相关性肿瘤细胞。结论：以控制EB病毒感染的疫苗致敏DC，能促进抗原提呈并启动抗肿瘤免疫，体外可有效杀伤EB病毒相关性肿瘤细胞。DC疫苗作为一种针对EB病毒相关性肿瘤的新型治疗性疫苗，会有着良好的研究价值和开发前景。     

EBNA1阳性肿瘤细胞株的建立

目的：建立表达Epstein-Barr(EB)病毒核抗原-l(EBNA1)的肿瘤细胞株，用于EB病毒相关肿瘤的研究。方法：将1200bp的EBNA1基因片段插入真核细胞表达载体pcDNA3中，使用脂质体介导pcDNA3／EBNA1质粒进入人鼻咽癌CNE2细胞和人宫颈癌Hela细胞，通过G418进行阳性克隆的筛选，PCR、RT-PCR进行鉴定。结果：成功建立EBNA1基因阳性的鼻咽癌和宫颈癌肿瘤细胞株，经PCR、RT-PCR检测证明两种转基因细胞株稳定表达EBNA1。结论：EBNA1阳性肿瘤细胞株的建立，为体外进行EB病毒相关肿瘤的研究提供细胞模型：     

EB病毒核抗原的生物学特性及其作用研究的进展

某些人类癌瘤(鼻咽癌,Burkitt瘤)细胞中存在着EB病毒基因组,而患者血清中对该病毒的抗体增加。EB病毒(EBV)又能诱发狨猴恶性淋巴瘤。因此,EB病毒对鼻咽癌的病因学作用引起了广泛的重视,在EB病毒与宿主细胞相互关系的研究中,抗原表达     

血清EB病毒VCA-IgA抗体测定对鼻咽癌诊断及监护病情的价值

鼻咽癌细胞中存在EB病毒核抗原和EB病毒基因组,提示鼻咽癌与EB病毒密切相关。研究证明,鼻咽癌患者血清中有抗EB病毒相关抗原的抗体升高,其中尤以抗EB病毒壳抗原(VCA)-IgA抗体升高最为特征。我们采用间接免疫荧光法测定了鼻咽癌患者及对照者血清中VCA-IgA抗体,观察了对鼻咽癌诊断及监护病情的价值。     

微量元素亚硒酸钠与硫酸镍对Epstein—Barr病毒壳抗原表达影响的研究

本文报告应用间接免疫荧光技术,研究了亚硒酸钠与硫酸镍对EB病毒壳抗原(VCA)表达的影响及其相互关系,0.1-1.0μg/ml亚硒酸钠1-20μg/ml硫酸镍,各自对VCA表达呈抑制和促进的相反效应。适当浓度的亚硒酸钠不仅能抵消各剂量硫酸镍对病毒抗原表达的促进作用,而且还明显抑制抗原自身的表达。时经镍预处理24小时后的靶细胞,硒仍显示出强烈的抑制效应:外一方面,硒一次预处理后,仍保持其对镍激发抗原能力的抑制。这些结果提示,硒对EBV-VCA表达的抑制作用远较镍的促进作用为强,而且持久。 本文还讨论了鼻咽癌患者体内的高镍低硒状态与EBV感染的相互关系及其可能在鼻咽癌发生、发展中所起的作用。     

Epstein-Barr virus-induced malignant lymphoma in a white-lipped marmoset

One of six white-lipped marmosets inoculated with cell-free B95-8 virus developed diffuse malignant lymphoma. Epstein-Barr virus (EBV) DNA was detected in a pathologically enlarged mandibular lymph node by DNA-DNA hybridization. The affected animal at the time of killing had EBV antibody titers of 1:320 for viral capsid antigen (VCA) and 1:20 for early antigen (EA) while all non-diseased animals had less than or equal to 1:80 VCA antibody and no detectable EA antibody. This is the first report of lymphoma development in a white-lipped marmoset following EBV inoculation.     

Effect of nickel sulfate on cellular proliferation and Epstein-Barr virus antigen expression in lymphoblastoid cell lines

Nickel is found in high levels in the environment of the high-risk areas for nasopharyngeal carcinoma (NPC) of China. Epstein-Barr virus (EBV) is associated with NPC and the interaction of nickel and EBV may be a contributive cofactor to the development of NPC. The study of the in vitro effect of nickel sulfate on cell proliferation and EBV-antigen expression demonstrated that nickel increases cell proliferation of some EBV-positive lymphoblastoid cell lines and increases early antigen expression of Raji cells. Nickel exerted variable effects on viral capsid antigen (VCA): increasing VCA-positive cells in B95-8 cells while decreasing VCA in P3HR-1 cells. It is proposed that the uptake of nickel in NPC high-risk areas could be one of the factors responsible for cancer development in the nasopharynx in China.     

Establishment of EBNA-expressing cell lines by infection of Epstein-Barr virus (EBV)-genome-negative human lymphoma cells with different EBV strains.

Cells of two EBNA (Epstein-Barr virus nuclear antigen)-negative human lymphoma cell lines, BJAB and RAMOS, were infected with two strains of Epstein-Barr virus (EBV). In two different experiments, B95-8 virus-infected BJAB cells revealed a gradually increasing number of EBNA-positive cells. Twenty weeks after infection almost 100% of the cell population expressed this antigen. In contrast, it has not so far been possible to convert RAMOS cells into an EBNA-positive cell line. The initial proportion of 35% EBNA-positive cells declined to about 10% 20 weeks after infection. The development of EBNA-positive multinuclear giant cells was a characteristic feature of infection with B95-8 virus. EA (early antigen) and VCA (virus capsid antigen) appeared in less than 0.1% of the cell population after induction with IUdR only. Infection of BJAB and RAMOS cells with P3HR-1 virus finally resulted in both cases in EBNA-positive lines. In contrast to B95-8 virus, the number of EBNA-positive lines. In contrast to B95-8 virus, the number of EBNA-positive cells remained below 1% during the first 6 to 8 weeks. A sudden increase occurred thereafter, bringing the number of EBNA-expressing cells to almost 100% within the following 4 weeks. During this period, BJAB but not RAMOS cells revealed a small number of EA- as well as VCA-positive cells (less than 0.1%). Thus, reinfection by spontaneously released virus may explain the sudden increase in EBNA-positive BJAB cells. Two distinct patterns of EBNA staining in P3HR-1 virus-infected cells were observed. They may suggest a genetic heterogeneity of this virus preparation.     

Differential induction of Epstein-Barr virus-related antigens in human lymphoblastoid cells by virus-mediated cell-to-cell contact.

Differential induction of Epstein-Barr virus (EBV)-related antigens was observed after Sendai virus-medicated fusion of producer and non-producer cells with various human and mouse cells. The EBV-determined early nuclear antigen (ENA), early antigen (EA) and viral capsid antigen (VCA) could be induced at a high rate in producer cells (P3HR-1 and B95-8), normally expressing these antigens at very low frequency, as early as 12-24 h after fusion with each other or with human amnion FL cells. In contrast, only one antigen, ENA, was induced in producer cells following fusion with non-producer cells. This limited induction occurred also in non-producer cells fused with FL cells, but not after fusion with each other. EBV induction did not result from fusion of either producer or non-producer cells with mouse cells (L-M (TK-) Cl1D and MCB-L). The differential induction was not the result of heterokaryon formation as determined by autoradiography. The implications of these findings are discussed.     

The transforming prototype of Epstein-Barr virus (B95-8) is also a lytic virus

The B95-8 isolate of the Epstein-Barr virus (EBV) has been described as a non-lytic transforming virus. We have performed experiments in order to determine if the B95-8 EBV is capable of super-infecting and replicating in EBV-genome-positive non-producer lymphoblastoid cells. Using concentrates of B95-8 EBV, prepared from 6 different B95-8 cell lines treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), we demonstrated that virus concentrates could transform human or cotton-top tamarin B-lymphocytes and also lytically replicate in Raji cells, inducing EBV antigens and infectious virus. While the virus obtained from B95-8 super-infected Raji cells was able to transform cord-blood lymphocytes (CBLs) and super-infect Raji cells, transformation was abortive, with cell cultures only growing for up to 6 weeks. Transformation titers of the B95-8 virus concentrates ranged from 10(5) to greater than 10(8) transforming units/ml; early antigen (EA) induction ranged from 1% to 50% after superinfection of Raji cells, depending on the virus stock used, as determined by immunofluorescence. Southern blot analysis was carried out on the DNA prepared from B95-8 cells and virion DNA. The results were consistent with the published EcoRI restriction pattern for B95-8 EBV. The issue of whether the B95-8 cells produce virions with a dual biological phenotype or, rather, 2 biologically distinct viruses, is addressed.     

Establishment and characterization of two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus and derived from nasopharyngeal carcinomas

Two epithelial tumor cell lines were established from biopsy specimens of 2 nasopharyngeal carcinomas (NPC) and designated HNE-1 and HONE-1. Uncloned HNE-1 cells were found to be Epstein-Barr virus (EBV) DNA-positive when examined by Southern blot analysis up to passage 35, after which the EBV genome could no longer be detected. A similar loss of EBV DNA took place in uncloned HONE-1 cells. However, HONE-1 clone 40 cells are still EBV DNA-positive up to passage 42 thus far and cell cultures contain 85-90% EBV nuclear antigen (EBNA)-positive cells. The HNE-1 cell line has been passaged more than 100 times and the uncloned HONE-1 cells more than 90 times. The tumorigenicity of the HNE-1 and HONE-1 cells was demonstrated by tumor induction in nude mice. Karyotypic analysis of the HNE-1 cells demonstrated an aneuploidy with a modal chromosomal number of 74 at passages 5 and 101 at passage 20; 18 marker chromosomes were identified. We have continued to map the EBV genome latently associated with the HNE-1 and HONE-1 cells using the Bam HI, EcoRI or Hind III restriction enzymes. Using EcoRI fragments A-K as probes, we found that HNE-1 EBV DNA is different from B95-8 and HR-1 EBV DNA in the EcoRI-C region. The Bam HI map for HONE-1 EBV DNA is very similar to the B95-8 map; it contains the Bam HI-Y fragment but without Bam HI B' and WI'. Differences were observed between HONE-1 EBV DNA and B95-8 DNA using the Hind III restriction enzyme. There was no evidence of spontaneous expression of the latent EBV genome in HNE-1 cells, and attempts to induce replication of the latent EBV genome and rescue infectious virus have failed, suggesting a tightly restricted virus genome.     

Characteristics of cell lines derived from human leukocytes transformed by different strains of Epstein-Barr virus.

Variation of Epstein-Barr virus (EBV) in respect to its effect on the properties of transformed cells was probed. Human umbilical cord leukocytes from six different individuals were transformed in vitro by either B95-8 (B) or QIMR-WIL (Q) strains of EBV and subsequently 12 lymphoblastoid cell lines (six B-derived and six Q-derived lines) were established. The B lines and Q lines were different in the expression of EBV genome i.e. production of virus or viral antigens, and in other properties including growth pattern and immunoglobulin production. The most striking differences between the two groups lay in their capacity to produce infectious virus and in the shape of the cell-clumps. The results suggest that different strains of EBV may induce transformed cells with different characteristics.     

Superinfection of epithelial hybrid cells (D98/HR-1, NPC-KT, and A2L/AH) with Epstein-Barr virus and the relationship to the C3d receptor

Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid cell lines (D98/HR-1, NPC-KT, and A2L/AH) were superinfected with EBV derived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1 virus is lytic and induces early antigen in superinfected Raji cells; the virus is not capable of transforming B-lymphocytes. Virus preparations from NPC-KT cells have both transforming and early antigen-inducing properties, while B95-8 virus can only transform B-lymphocytes. It was possible to demonstrate EBV antigens after superinfection of D98/HR-1 cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells, which were originally prepared by fusing human adenoid epithelial cells (Ad-AH) and EBV genome-positive NPC explanted epithelial cells, were susceptible to superinfection with HR-1 virus but not to NPC-EBV. The A2L/AH hybrid cells, which were prepared by fusion between Ad-AH cells and lymphocytes transformed by B95-8 virus, could not be superinfected with any of the virus preparations. In order to further investigate the nature of the EBV receptor as it relates to other cell membrane components, we examined cell surface markers on Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 cells using monoclonal antibodies and by rosette formation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines express the OKB2 antigen and that the common acute lymphoblastic leukemia antigen is expressed on the A2L/AH cells. We also found that NPC-KT parental cells and a clone of NPC-KT cells express erythrocyte antibody complement b and erythrocyte antibody complement d, as determined by rosette formation, but were negative for C3b and C3d when monoclonal antibodies against these two markers were used. The D98/HR-1 cells were also confirmed to be negative for C3b and C3d. The data suggest that the C3d receptor may be part of the EBV receptor but that the C3d receptor, by itself, is not the only receptor to which EBV can bind.     

Epstein-Barr virus: transformation of non-human primate lymphocytes in vitro

Continuous lymphoblastoid cell cultures were established from marmoset (Saguinus sp.), squirrel (Saimiri sciureus), owl (Aotus trivirgatus) and cebus (Cebus apella) monkeys after culturing their peripheral lymphocytes with lethally X-irradiated cells carrying Epstein-Barr virus (EBV). Transformation also was achieved by exposing simian lymphocytes to infectious, cell-free EBV derived from the simian lymphoblastoid cell cultures. Simian lymphocytes were not transformed after exposure to cell-free EBV derived from HR-1 cells. The simian cell cultures were similar to cell cultures derived from Burkitt's lymphoma or infectious mononucleosis patients. EBV-induced early, viral capsid and membrane antigens, intranuclear inclusion bodies and herpesvirus virions were demonstrable in most cultures. Seven cultures were insusceptible to superinfection with EBV and treatment of the cultures with halogenated pyrimidines was relatively ineffective for inducing synthesis of early or viral capsid antigens. All cell cultures had B-cell characteristics: they produced immunoglobulins but did not form spontaneous rosettes with sheep erythrocytes. Four of six marmoset monkeys, inoculated with EBV-transformed marmoset lymphocytes, developed antibodies to EB viral capsid antigens and one marmoset inoculated with autochthonous transformed cells also developed heterophile antibodies. Seven marmosets, inoculated with cell-free EBV derived from HR-1 cell cultures, developed no detectable levels of antibodies to EBV-specified antigens or heterophile antibodies. No overt clinical abnormalities were detected in any of the marmosets inoculated with HR-1 or Kaplan EBV but one of five marmosets inoculated with B95hyphen;8 EBV developed a lymphoma.     

Abortive expression of the Epstein-Barr virus (EBV) cycle in a variety of EBV DNA-containing cell lines, as reflected by nucleic acid hybridization in situ.

A variety of Epstein-Barr virus (EBV) DNA-containing cell lines have been tested for the expression of the EBV-associated antigens EBNA (nuclear antigen), EA (early antigen), and VCA (viral capsid antigen), and for the presence of cells containing disproportionate amounts of EBV DNA. The antigen tests utilized immunofluorescence and 125I-labelled antibodies combined with autoradiography. EBV-DNA was detected by in situ hybridization with 3H-labelled EBV RNA complementary to P3HR-1 EBV DNA (P-EBVcRNA). The P-EBVcRNA has been shown to represent the majority of the P3HR-1 EBV DNA sequences. It was concluded that EBV DNA-containing cell lines can be divided into those that express only EBNA, those that express EBNA and EA and those that express EBNA, EA and VCA and also contain cells that undergo disproportionate EBV DNA synthesis. Consequently, in some cell lines there is an abortive expression of the EBV cycle in that some cells spontaneously express EA but fail to continue further to viral DNA synthesis. A similar pattern can be found after experimental induction of the EBV cycle, suggesting that related mechanisms govern the spontaneous expression of the EBV cycle and the extent of its inducibility.