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。     

几种药物抑制EB病毒壳抗原在体外细胞中表达的作用

ＥＢＶ和ＮＰＣ有密切的关系，用三氮唑核苷、聚肌胞、柴胡、鱼金、板兰根、抗毒清咽合剂按不同浓度加入到能自发产生ＥＢＶ的Ｂ９５－８细胞悬液中，这几种药物均有一定的抑制细胞中ＥＢＶ－ＶＣＡ表达的能力，其中三氮唑核苷（１０μｇ／ｍｌ）、板兰根（１０ｍｇ／ｍｌ）和抗毒清咽合剂（１：１０００）则有较强的抗ＥＢＶ作用，抑制率分别达到了５５．５６％、５７．８９％和５４．１７％，而聚肌胞、柴胡、鱼金的抑制作用较弱。实验结果表明，用药物抑制ＥＢＶ的复制和表达，有可能作为控制ＮＰＣ的方法之一。     

几种药物抑制EB病毒壳原在体外细胞中表达的作用

ＥＢＶ和ＮＰＣ有密切的关系，用三氮唑核苷，聚肌胞，柴胡，鱼金，板兰根，抗毒清咽合剂按不同浓度加入到能自发产生ＥＢＶ的Ｂ９５－８细胞悬液中，这几种药物均有一定的抑制细胞中ＥＢＶ－ＶＣＡ表达的能力，其中三氮唑核苷（１０μｇ／ｍｌ），板兰根（１０ｍｇ／ｍｌ）和抗毒清咽合剂（１：１０００）则有较强的抗ＥＢＶ作用，抑制率分别达到了５５．５６％，５７．８９％和５４．１７％，而聚肌胞，柴胡，鱼金的抑制作用较弱，     

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。     

V-val突变型EB病毒核抗原1的功能研究

背景与目的:EB病毒核抗原1(Epstein-Barr viral nuclear antigen 1,EBNA 1)对于维持EB病毒的潜伏感染有重要作用。V-val变异型的EBNA1与鼻咽癌有密切的相关性。本研究旨在比较原型和V-val变异型的EBNA1基因在上皮细胞中的功能的差异。方法:用PCR方法扩增出原型和V-val变异型EBNA1基因的全长并克隆到pGFP载体上,转染HEK293细胞,检测两种亚型的EBNA1蛋白的表达对细胞生物学性状的影响。以含有EB病毒增强子FR序列的荧光素酶载体作为报告基因,比较两种亚型的EBNA1基因对质粒的转录激活能力。结果:原型和V-val变异型EBNA1基因的表达对HEK293细胞的生长速度没有明显影响,但表达原型EBNA1的细胞的克隆形成能力明显低于V-val亚型。在裸鼠致瘤实验中,接种表达原型和V-val变异型EBNA1细胞的实验组均未见肿瘤形成。但是在瞬时转染实验中,表达V-val变异型EBNA1基因的HEK293细胞的荧光素酶活性明显高于原型EBNA1基因。结论:原型和变异型EBNA1均未发现有明显的转化细胞的能力,但是V-val变异型EBNA1蛋白与原型相比,其转录激活的功能明显增强。     

EB病毒诱导的膜抗原（MA）及其抗体的研究

近年来,EB病毒诱导的膜抗原及抗体研究受到重视,主要基于二个原因:(1)膜抗原的有效成份已被提取,且可诱导机体产生抗病毒中和抗体。因此膜抗原的抗体水平可作为EB病毒疫苗接种效果的监测指标。(2)在多种EB病毒相关抗体中,MA抗体诊断鼻咽癌具有较高的特异性。作者对EB病毒诱导的膜抗原及抗体的研究作一综述,着重MA抗体的检测及意义。     

基因工程膜抗原检测EB病毒MA—IgA抗体研究及与VCA—IgA...

With gene engineering EB virus membrane antigen as the diagnostic antigen, indirect immunofluorescence (IF) assay was used to detect IgA antibody against EB virus membrane antigen (MA-IgA) in sera from 202 nasopharyngeal carcinoma (NPC) patients and 315 controls (normal and patients with other tumors). MA-IgA antibody was positive in 96.8% of the pre-treatment NPC patients with a GMT of 1:36.3. MA-IgA detection by this method was more sensitive than EA-IgA detection by IE. In contrast, patients with tumors other than NPC were negative for MA-IgA antibody. 9.1% of VCA-IgA positive persons were MA-IgA positive with a GMT of less than 1:5. No MA-IgA positive was found in VCA-IgA negatives. The results indicated that this method was relatively specific. In the treatment group, the positive rate and GMT of MA-IgA antibody declined with increase in survival time and the decline was faster than VCA-IgA. When recurrence or distant metastasis developed, similar to VCA-IgA and EA-IgA antibodies, the positive rate and GMT of MA-IgA antibody increased to its pretreatment level. Therefore, MA-IgA detection might be valuable in the early diagnosis and monitor of NPC.     

鼻咽癌患者亲属血清抗EB病毒壳抗原抗体水平的探讨

作者检测鼻咽癌患者亲属397人的血清 EB 病毒 IgA/VCA 抗体水平,并与正常人群作比较。结果显示(1)两组间抗体水平无显著差异;(2)在患者的子女、兄弟姐妹和夫妻之间,各组相互比较也无差异。因此作者认为在鼻咽癌的发生过程中,除 EB 病毒外,还有遗传或(和)环境因素的介入。     

抗重组EB病毒抗原双重抗体检测血清学诊断鼻咽癌的研究

背景与目的：在评估4种EB病毒抗原酶联免疫吸附法的基础上,探讨优化抗重组：EB病毒抗原双重抗体检测应用于血清学诊断鼻咽癌。方法：收集广州地区57例治疗前鼻咽癌患者和58例健康成人的血清。应用：EB病毒特异抗原(谷胱甘肽转移酶重组融合蛋白)为基础的4种免疫酶联吸附法,即：EBNA1-IgA,EBNA1-Igg,Zta-IgA和Zta-IgG检测血清中抗EB病毒的抗体水平。结果：EBNA1-IgA的灵敏度(O．9123)和阴性预测值(0．9074)是单独使用4种ELISA实验中最高的。Zta-IgA具有最高的正确率(π,0．8870)和Youden指数(J,0．7738)。当评估配对的ELISA时,EBNA1-IgA和Zta-IgA双重阳性的所有指标是4种双重阳性实验中最高的。5例：EBNA1-IgA阴性的鼻咽癌患者呈Zta-IgA阳性,而7例Zta-IgA阴性的鼻咽癌患者呈EBNA1-IgA阳性。结论：EBNA1-IgA酶联免疫吸附的单独检测在血清学诊断鼻咽癌时优于其他3项(EBNA1-IgG、Zta-IgA和Zta-IgG)单独酶联免疫吸附检测。EBNA1-IgA和Zta-IgA两项的组合应用在血清学诊断鼻咽癌时有互补作用,是血清学检测的合适组合。     

Morphological transformation of nasopharyngeal epithelial cells in vitro by Epstein-Barr virus from B95-8 cells.

Tissue fragments of fresh biopsy specimens from the non-neoplastic NP mucosa of 20 subjects, from the tumours of 10 NPC patients, from the mucosa of freshly removed tonsils from nine subjects and from the primary lesions of seven patients with malignancies of the upper respiratory or alimentary tract other than NPC were infected with EBV derived from B95-8 cells. A significantly higher frequency of growth stimulation and a greater mean growth ratio between infected and uninfected fragments from the same source were observed in the non-neoplastic NP mucosa specimens than in the others. In addition, the growth characteristics and the morphology of the cells in the infected non-neoplastic NP mucosal explants resembled those of transformed cells; but whether they possess malignant potential is being investigated.     

Significance of Plasma IgA and IgG Antibodies to Epstein-Barr Virus Early and Viral Capsid Antigens in Thai Nasopharyngeal Carcinoma

Epstein-Barr virus (EBV) is an important causal factor of human nasopharyngeal carcinoma (NPC). High levels ‍of serum IgA and IgG antibodies to EBV early and viral capsid antigens (IgA/EA, IgA/VCA, IgG/EA and IgG/VCA) ‍have been reported in NPC patients. Since specific serum IgA/EA, IgA/VCA and IgG/EA are claimed to be useful ‍serological markers for NPC. In order to evaluate whether plasma IgA/EA, IgA/VCA, IgG/EA and IgG/VCA antibody ‍levels are useful markers for diagnosis and prognosis of Thai NPC, we examined the prevalence of these antibodies ‍in 79 NPC patients, and 127 age-matched controls (47 healthy subjects (HS), 32 cases of other disease (OD) and 48 ‍cases of other cancer (OC)) by using an indirect immunofluorescence assay. The prevalence of plasma IgA/EA, IgA/ ‍VCA, and IgG/EA in NPC patients (55.7, 68.4 and 68.4%) was significantly higher than in the HS (0.0, 0.0 and ‍20.5%,), OD (0.0, 0.0 and 3.1%) and OC (0.0, 0.0 and 20.8%) groups (p<0.05). The prevalence of plasma IgG/VCA ‍in NPC patients (93.7%) was significantly different from those for the OD and OC groups (71.9 and 43.8%) but not ‍for the HS group (89.4%). In NPC patients, the geometric mean titers (GMT) of plasma IgA/EA, IgA/VCA and IgG/ ‍EA were increased with an advanced clinical stage of disease but not IgG/VCA. In contrast, GMT of IgG/VCA was ‍increased with aggressive type of disease (histological type) but not IgA/EA, IgA/VCA, and IgG/VCA. The results of ‍our study suggest that plasma IgA/EA, IgA/VCA and IgG/EA antibodies may be useful markers for diagnosis and ‍assessing prognosis of Thai NPC. ‍     

Possible transformation of nasopharyngeal epithelial cells in culture with Epstein-Barr virus from B95-8 cells.

Explants of fresh biopsy specimens from non-neoplastic nasopharyngeal (NP) mucosa, nasopharyngeal carcinoma (NPC), other tumours (OT) of the head and neck and freshly removed tonsils were treated with an Epstein-Barr virus (EBV) preparation from B95-8 cells and cultured. The mainly epitheloid outgrowths from these infected explants were then compared with those from their respective uninfected controls at 14 days. Growth stimulation occurred with a significantly higher frequency, and the degree of stimulation was generally higher with the infected NP explants than those of the similarly infected explants of other origins. Furthermore, after treatment with the virus preparation, several of the outgrowths from the NP explants showed growth characteristics and cellular morphology typical of those of transformed cells. Light microscopy has shown the changed NP cells to have epithelial characteristics. This is now being verified by electron microscopy, which has so far shown the presence of keratin fibrils and desmosomes in one specimen examined. They are also being examined for the presence of EBV-DNA and EBNA, and other features of transformation, including malignant tendency, by passage through athymic nude mice.     

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.     

Demethylation by 5-Azacytidine Results in the Expression of Hepatitis B Virus Surface Antigen in Transgenic Mice

In 14p3HB transgenic mice, which carry three tandem copies of hepatitis B virus (HBV) DNA, the HBV DNA was significantly methylated and no viral proteins were produced. To analyze the causal relationship between hypermethylation and gene inactivity, 5-azacytidine was injected into the mice to demethylate HBV DNA. When postnatal 14p3HB mice were treated with the drug, hepatitis virus surface antigen was produced in these mice by 3 weeks of age, and the integrated HBV DNA of the liver was less heavily methylated. Our results suggest that injection of 5-azacytidine can be used to efficiently activate a silent transgene such as HBV DNA in transgenic mice.     

多胺对Raji细胞中Epstein-Barr病毒早期抗原的诱导

目的 探讨精胺和亚精胺对EB病毒抗原的诱导作用。方法 分别利用细胞计数法和免疫酶法对Raji细胞进行活细胞计数和500个细胞中有Epstein-Barr病毒早期抗原的表达细胞数。结果 精胺和亚精胺的半数致死量分别为970.4094ng/ml和87.71987ng/ml；精胺和亚精胺单独诱导EB早期抗原表达率分别为4．6％和4．2％；而精胺，亚精胺与正丁酸协同作用时，其早期抗原表达率分别达到20．6％，21．6％。结论 精胺和亚精胺可诱导Raji细胞内Epstein-Barr病毒早期抗原的表达，当它们分别与正丁酸协同作用时，其表达率增高。     

EB病毒IgA/VCA阳性的鼻咽癌非病毒危险因素的病例-对照研究

目的 以EB病毒IgA/VCA阳性为入列条件,进行大样本的病例 对照研究,系统性地评估鼻咽癌家族史,环境因素包括饮食、吸烟和职业暴露在中国南方鼻咽癌发病中的作用。方法 选择的1 010例经病理学确诊的鼻咽癌病人及1 009例正常人对照均来源于梧州市和苍梧县及其周边广西和广东高发市县。病例与对照的EB病毒IgA/VCA血清学检测均为阳性。对有关暴露因素进行单因素及多因素非条件Logistic回归分析。结果 研究结果显示有鼻咽癌一级亲属的EB病毒IgA/VCA阳性个体患鼻咽癌的风险会升高3.05倍。食用咸鱼、经常接触柴火及有机溶剂、较早年龄开始吸烟会显著增加患鼻咽癌的风险,其OR值分别为1.70、4.04、2.23、1.68。结论 在调整EB病毒感染状态因素后,研究结果证实在南方高发区,鼻咽癌家族史、食用咸鱼、接触柴火及有机溶剂、吸烟仍然是鼻咽癌的非病毒危险因素。     

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。     

Detection of Epstein-Barr Virus DNA in Hodgkin- and Reed-Sternberg-Cells by Single Cell PCR

The Epstein-Barr virus (EBV) can be detected in the majority of lymph nodes involved by Hodgkin's lymphoma using the highly sensitive polymerase chain reaction (PCR). However, the rate of EBV-DNA detection by in-situ hybridisation, which allows allocation of EBV to a defined cell population, i.e. the neoplastic H&RS-cells, is lower. In an attempt to combine the advantages of the high sensitivity of the PCR and the possibility of cellular allocation by in-situ hybridisation, we established a single-cell PCR of Hodgkin- and Reed-Stemberg (H&RS)-cells isolated by micromanipulation from biopsy tissues. We amplified EBV sequences from the BamW-region by single-cell PCR. Using this method we were able to detect EBV-DNA in the H&RS-cells from 4 of 6 patients. In EBV positive cases all H&RS-cells of a given patient were positive, proving the high sensitivity and reproducibility of the method. Other cells in the biopsy tissue involved by EBV-positive H&RS-cells were shown to be negative. This indicates that EBV may have a role in the pathogenesis of many but not all cases of Hodgkin's disease.