检测马疱疹病毒4型抗体间接ELISA方法的建立及应用

为建立检测马疱疹病毒4型(EHV4)抗体的间接ELISA方法,本研究以原核表达的具有型特异性抗原表位的gG蛋白为检测抗原,经条件优化,成功建立检测EHV4抗体的间接ELISA方法,对该方法进行特异性及重复性试验。结果表明,重组4型gG蛋白仅与EHV4阳性血清发生反应,不与马疱疹病毒1型(EHV1)阳性血清发生反应;组内及组间变异系数均小于10%。采用建立的方法与进口试剂盒对不同省份送检的马血清进行检测比较,两者阳性和阴性符合率均在90%以上。本研究建立的间接ELISA方法为检测EHV4抗体及流行病学调查奠定基础。

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

A case of rapid growing colonic NK/T cell lymphoma complicated by Crohn's disease

A 37-year-old man developed abdominal pain and bloody diarrhea 11 months before admission. The colonoscopy revealed multifocal ulcers in the colon. Histology showed active chronic inflammation. Although anti-tuberculosis medication was effective, his symptoms repeated 2 months later. The subsequent colonoscopy revealed more extensive irregular ulcers than before, and he was clinically suspected with intestinal malignant lymphoma. He underwent subtotal colectomy and was histologically suggested Crohn＇s disease, then 5-aminosalicylic and a combination of prednisone and azathioprine were administered in succession postoperatively, but they achieved minimal relief of symptoms for a period of 7 months. The third colonoscopy showed a large irregular ulcer in the ileocolon stomas, and primary colonic NK/T cell lymphoma was diagnosed through histological and immunophenotypic studies. Malignant lymphoma should be taken into consideration when clinically diagnosed Crohn＇s disease was refractory to medication or when its clinical course became aggressive.

鼻咽癌病因学和防治研究

鼻咽癌是我国南方地区常见的恶性肿瘤,鼻咽癌具有显著的地区聚集性,大量的流行病学调查资料表明,鼻咽癌发生与EB病毒的流行,家族遗传因素及环境因素有关,鼻咽癌的发生是多基因、多因素和多阶段交互作用的结果。通过检测鼻咽癌患者血液中各种与EB病毒相关的因子,可以发现早期鼻咽癌患者,探讨其与转移和预后的关系。

Epstein-Barr virus induces human nasopharyngeal epithelial cells to escape from the replicative senescence

OBJECTIVE: To observe the biological changes of primary human nasopharyngeal epithelial cells in the early stage of immortalization. METHODS: The morphological changes of nasopharyngeal epithelial cells were observed by phase contrast microscopy, and the activity profile of senescence-associated beta-galactosidase (SA-beta-Gal) was detected by SA-beta-Gal staining. The expression of p16(INK4a) protein was tested by immunochemical assay, and the life span in vitro of nasopharyngeal epithelial cells was calculated as population doublings. In addition, the expression of Epstein-Barr (EB) virus latent membrane protein 1 (LMP1) was also detected by immunofluorescence staining. RESULTS: Morphologically, cells treated with EB virus and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) formed multi-layer foci, and their cellular life span in vitro was extended (about 155 days of culture). A low percentage of cells (about 4.8%) expressed SA-beta-Gal activity at late primary culture, and did not always express p16(INK4a) protein in the progression of culture. CONCLUSIONS: Nasopharyngeal epithelial cells treated with EB virus in cooperation with TPA can pass through the stage of senescence and enter the early stage of immortalization. Some changes of phenotype occur in these cells.Our results provide data for further studying the mechanism of immortalization and the establishment of a human nasopharyngeal epithelial cell line.

Detection of Epstein-Barr virus DNA in plasma/serum:a useful serological indicator for diagnosis of nasopharyngeal carcinoma

OBJECTIVE: To compare the detection rates of Epstein-Barr virus (EBV) DNA in the serum/plasma between apparently healthy adults (AHAs) and nasopharyngeal carcinoma (NPC) patients in attempt to evaluate the efficiency of EBV DNA assay for serodiagnosis of NPC. METHODS: The plasma and serum were obtained from 58 AHAs and 66 untreated NPC patients. EBV DNA W-fragment was detected using nested ploymerase chain reaction (PCR). Immunoenzymatic assay for titration of IgA-VCA was also adopted. RESULTS: EBV DNA detection rate (84.85%) in the plasma/serum of 66 NPC patients was significantly higher than that (10.34%) in 58 AHAs. The sensitivity of plasma/serum EBV DNA assay (0.8485) was higher than that (0.8030) of titrating IgA-VCA (positive criterion >/= 1:40) though the specificities of these two tests were the same (0.8966). The correct rate, predictive value of a positive test, and Odds ratio of dual positivity (0.8387, 0.9792 and 141.0, respectively) were higher than those of single positivity either to plasma/serum EBV assay (0.5242, 0.7333 and 1.1423, respectively) or to IgA-VCA >/= 1:40 test (0.4839, 0.5385 and 1.0480, respectively). CONCLUSION: The EBV DNA detection in the plasma/serum using nested PCR may be a useful indicator for serodiagnosis of NPC.

三株人鼻咽癌Scid小鼠移植瘤的建立及特性研究

目的 建立鼻咽癌鼠移植瘤 ,为鼻咽癌的特异性免疫治疗研究提供实验模型。方法 从 2 6例未治的鼻咽癌病人鼻咽部铰取肿瘤组织 ,分别皮下接种BALB c裸小鼠 (14只 )和Scid小鼠 (12只 ) ;成功建立移植瘤株后观察其生长特性、形态、染色体和LMP的表达情况。结果 成功建立鼻咽癌Scid小鼠移植瘤株CSNET 1、CSNET 2和CSNET 3,已分别传至第 11代 (2 3个月 )、第 14代 (17个月 )和第 9代 (16个月 ) ,其光镜和电镜下形态符合低分化鳞癌的特征 ,电镜下在CSNET 1中可观察到Ep stein Barr病毒样颗粒 ,CSNET 1的染色体全部为人体细胞来源的染色体 ,免疫组化检测结果 ,3个瘤株均可见EB病毒晚期膜蛋白LMP 1和LMP 2阳性细胞。结论 CSNET 1、CSNET 2和CSNET 3是人类来源的鼠移植瘤株 。

马鼻肺炎病毒双重PCR分型检测方法的建立

根据GenBank中登录的马鼻肺炎病毒1、4型(EHV1、EHV4)糖蛋白B(gB)基因特异保守序列(登录号分别为AY665713、AF030027.1),各设计1对引物,建立了同时分型检测EHV1、EHV4的双重PCR方法;确定其最佳工作条件,并对该方法进行了特异性和敏感性试验。结果显示,在EHV1、EHV4中分别扩增出265bp和537bp的特异性目的条带,而马流感病毒cDNA、马动脉炎病毒cDNA、马乙型脑炎病毒cDNA及马传染性贫血弱毒疫苗株cDNA均未扩增出任何条带。EHV1的DNA最低检出限为2.1pg,EHV4的最低检出限为15pg。表明本试验建立的方法具备很高的应用价值。

Epstein-Barr病毒bhrfl表达对鼻咽癌细胞株CNE2拓扑异构酶Ⅰ表达的影响

目的 了解Epstein Barr(EB)病毒基因bhrfl表达对鼻咽癌细胞拓扑异构酶Ⅰ表达的影响。方法 通过构建bhrfl表达载体并转染鼻咽癌细胞株CNE2细胞 ,以检测癌细胞在转染后其拓扑异构酶Ⅰ表达及其抗喜树碱诱发凋亡能力的改变。结果 bhrfl表达可增强癌细胞拓扑异构酶Ⅰ的表达 ,使癌细胞对喜树碱的敏感性下降 ,生存时间延长 ,凋亡率减少 (P <0 0 1)。结论 bhrfl的表达可通过增强CNE2细胞拓扑异构酶Ⅰ的表达而增强其抗喜树碱诱发的细胞凋亡作用。

EB病毒潜伏膜蛋白1与鼻咽癌

鼻咽癌（nasopharyngeal carcinoma,NPC）为我国南方高发的上皮来源的肿瘤,其病因不清。大量研究表明,EB病毒（Epstein-Barr virus,EBV）编码的潜伏膜蛋白1（latent mem- brane protein 1,LMP1）是目前唯一被证实与细胞转化有关的具有癌基因功能的潜伏膜蛋白,通过干扰细胞内信号传导途径等发挥作用,促进鼻咽癌发生、发展和转移。本文旨在对EB病毒LMP1与鼻咽癌的发生发展研究现状做一综述。

EB病毒编码潜伏膜蛋白－1与鼻咽癌免疫治疗

综述近年来EB病毒（Epstein—Barrin virus）编码的潜伏膜蛋白1（Iatent membrane protein，LMP－1）对鼻咽癌（nasopharyngeal carcinoma，NPC）患者免疫系统影响和对其进行免疫治疗方面的研究进展情况，并着重介绍针对潜伏膜蛋白1的免疫治疗研究现状。

荧光定量PCR技术及其在鼻咽癌中的研究状况

荧光定量PCR(fluorescencequantitativepolymerasechainreaction,FQ-PCR)是新研制出的一种核酸定量技术。该技术在PCR反应系统中引入了荧光标记探针,具有高灵敏性、高特异性和高准确性的特点。目前已被初步应用于鼻咽癌诊断、临床疗效判断、肿瘤复发及转移监测等多个方面。