EB病毒潜伏膜蛋白LMP1与P53在鼻咽癌中表达的意义

目的：探讨EB病毒潜伏感染和抑癌基因P53异常表达与鼻咽癌的关系。方法：用免疫组化S－P方法对59例鼻咽癌（NPC）和12例鼻咽部正常或慢性炎症组织进行单克隆抗体LMP1，P53标记，并结合组织学类型、分化程度，发病年龄进行分组研究。结果：正常和慢性炎症的鼻咽粘膜均呈阴性表达，NPC中，LMP1阳性率69．49％（41／59），P53阳性率44．06％（26／59），双阳性率30．51％（18／59），LMP1在非角化性癌，低分化癌及青年组NPC中高表达；在鳞癌，高分化癌及老年组中低表达，P53在低分化癌中高表达，但与NPC组织学类型，发病年龄不存在明显相关性。结论：在NPC发生、发展过程中EBV潜伏感染起主导作用，P53基因异常表达可能起协同作用，EB病毒潜伏感染是NPC发病年龄年轻化的主要原因之一。     

EGCG诱导鼻咽癌细胞凋亡的cDNA阵列分析

目的　探讨茶中多酚类物质EGCG介导鼻咽癌细胞凋亡的机制以及对EB病毒潜伏膜蛋白 (LMP1)诱导凋亡的干预作用。方法　采用已建立的受四环素调控的LMP1表达的鼻咽癌细胞系 (pTet on LMP1HNE2 ) ,经 0 6mg·L-1Dox和 10 0mg·L-1EGCG处理 2 4h后 ,抽提RNA ,分别与含有细胞凋亡相关基因为主的AtlasapoptosiscDNAexpres sionarray膜杂交。结果　EGCG上调或下调某些与鼻咽癌细胞凋亡相关基因表达。结论　EGCG诱导肿瘤细胞凋亡的作用是多个基因及多条信号转导通路作用的结果 ,并对LMP1诱导鼻咽癌细胞凋亡相关基因表达有一定的干预作用     

EB病毒膜潜伏蛋白LMP1和基质金属蛋白酶MMP9表达与鼻咽癌转移的关系

目的探讨EB病毒膜潜伏蛋白LMP1和基质金属蛋白酶MMP9在鼻咽癌组织表达的意义。方法采用免疫组织化学法对40例鼻咽癌组织进行标记及分析。结果鼻咽癌淋巴结转移组LMP1和MMP9阳性表达率明显高于无淋巴结转移组,二者阳性表达呈明显相关。结论 LMP1和MMP9的表达与鼻咽癌的浸润及转移密切相关,LMP1介导MMP9在鼻咽癌细胞扩散和转移起重要作用。     

贵州省苗族、布依族及汉族 EB病毒感染情况及鼻咽癌诊断试验效果评价研究

目的调查贵州省苗族、布依族及汉族EB病毒感染情况并探讨EB病毒特异性Zta-IgA、EBNA1-IgA与VCA-IgA抗体定量检测对鼻咽癌的诊断价值。方法采用随机抽样的方法抽取苗族、布依族及汉族共3040例研究对象,收集研究对象的基本信息;采用ELISA法检测研究对象血浆EB病毒的Zta-IgA、EBNA1-IgA、VCA-IgA标志物水平,分析上述指标诊断鼻咽癌的价值。结果3040例研究对象中,Zta-IgA阳性率为15．03％;EBNA1-IgA阳性率为16．09％;VCA-IgA阳性率为14．41％（χ2=16．027,P=0．000）;Zta=IgA、EBNAI-IgA、VCA-IgA阳性率均为布依族最高（Pd0．05）;〉45岁组Zta-IgA、EBNA1-IgA、VCA-IgA阳性率均明显高于≤45岁年龄组（P〈0．05）;吸烟、饮酒可以明显提高Zta-IgA、EBNA1-IgA、VCA-IgA阳性率（P〈0．05）;三者联合检测对诊断鼻咽癌的灵敏度和特异度均明显升高。结论贵州省少数民族EB感染率水平较高,应开展Zta-IgA、EB-NA1-IgA、VCA-IgA监测;三者联合检测对鼻咽癌的早监测、早发现有积极意义。     

贵州地区非免疫缺陷相关性B细胞淋巴瘤与EB病毒的关系

目的 探讨非免疫缺陷相关性B细胞淋巴瘤与EB病毒的关系。方法 采用原位杂交、免疫组织化学方法分别检测63例贵州地区非免疫缺陷相关性B细胞淋巴瘤中的EBER1／2、LMP－1蛋白。结果 63例B细胞淋巴瘤中,6例EBER1／2（＋）（9．5％）,其中有4／13例上呼吸道淋巴瘤（30．8％）;仅有1／63例（1．6％）LMP－1（＋）。结论 非免疫缺陷相关性B细胞淋巴瘤与EB病毒的相关性小,其致瘤作用的发挥可能与机体的免疫状态有关。     

重组人酸性成纤维细胞生长因子C-末端测序

目的：应用蛋白质N-末端序列分析仪测定重组人酸性成纤维细胞生长因子（aFGF）C-末端氨基酸序列。方法：应用多肽分析软件选择合适的蛋白内切酶完全酶切aFGF，酶切后用RP—HPLC进行分离，收集软件预测C-末端肽段保留时间处的肽段峰，用蛋白质N-末端序列分析仪直接测得该肽段氨基酸全序列。结果：实测肽图图谱与软件预测理论图谱一致，所得到的肽段为aFGF C-末端肽段，经测序其结果与理论序列完全一致。结论：通过选择合适的蛋白内切酶，可运用RP—HPLC和蛋白质N-末端序列分析仪测定基因工程产品C-末端氨基酸序列。     

bcl-2基因产物在EB病毒相关性T细胞淋巴瘤中的表达

目的：探讨抗凋亡基因产物blc－2与鼻咽部T-细胞淋巴瘤发生与EB病毒感染的关系。方法：应用双温PCR技术和免疫组化（SP法）检测18例鼻咽T-细胞淋巴瘤组织中EB病毒DNA和抗凋亡基因产物bcl-2的表达。结果：15例EB病毒DNA阳性病例中7例出现blc－2表达（46.67％）,3例EB病毒DNA阴性病例中有1例bcl-2呈阳性反应（33．33％）。两组间无显著差异（P＞0．05）。结论：鼻咽T细胞淋巴瘤发病可能与EBV感染有关。bcl－2基因产物在鼻咽T-细胞淋巴瘤中异常表达与EB病毒感染无明显关系。     

Borna病病毒与精神分裂症的关系研究

摘要目的探讨贵州省部分地区博尔纳病病毒（Bornadiseocevicus）在精神分裂症患者中的感染情况及与疾病发生的相关性。方法采用巢式逆转录荧光定量PCR法对来自于贵州省部分地区（遵义、毕节、铜仁）的30例精神分裂症（SCH）患者及100例健康人的外周血液单核细胞（PB—MC）中BDVp24基因片段进行检测,对阳性产物进行纯化、克隆、测序,用软件对所测定的序列结果进行同源性分析。结果精神分裂症患者中BDVp24基因片段阳性率为6．7％（2／30）;健康体检者中未检出,二者检出率比较无统计学差异（P〉0．05）。精神分裂症患者测序结果与GenBank提供的strainV毒株、C6BV毒株、He80／FR病毒株进行比较,突变率均为3％,均有3个位点出现一致性沉寂突变。与1766毒株进行比较,突变率为2％,有2个位点出现一致性沉寂突变,即与1766毒株的亲缘关系最近。结论本研究发现30例精神分裂症患者中检出BDVp24基因片段阳性2例,而健康人中未检出,说明Borna病病毒感染可能与精神分裂症有关,两组问BDV检出率无统计学差异,可能与所采标本地区较局限或标本量少有关。     

应用线粒体12srRNA和COX-1基因进行种属鉴定

目的以线粒体DNA为目标序列,探讨生物检材的种属来源问题。方法收集人和7种动物的血液或肌肉组织样本,提取DNA定量后,复合扩增线粒体12srRNA和COX-1基因片段,2%琼脂糖凝胶电泳检测扩增产物谱带。结果人类DNA扩增产物分别为436 bp、277 bp两条谱带,而鼠、鸡、猪、兔、猫、狗、羊的扩增产物仅有一条谱带,430～450 bp,即动物基于COX-1基因片段无扩增产物。结论复合扩增线粒体12srRNA和COX-1两基因片段进行种属鉴定,方法简单,灵敏度较高,可应用于法医学实践。     

人类白细胞抗原-DRB1基因启动子区序列多态性与肺结核的相关性分析

目的探讨中国北方地区人群人类白细胞抗原（HLA）-DRB1基因启动子区序列多态性与肺结核的相关性。方法北方汉族肺结核患者97例为肺结核组，北方汉族健康人62例为对照组，采用PCR-SSP法对2组的HLA-DRB1基因启动子区进行PCR扩增，启动子区扩增产物直接测序。结果肺结核组和对照组的HLA-DRB1基因启动子区序列完全一致；对照组启动子区序列与数据库检索的序列比较有3个突变点：-26位的T被C替代，-49位的C被G替代，-78位的C被G替代。结论中国北方地区人群HLA-DRB1基因启动子区序列具有多态性，但此多态性与肺结核的发病没有相关性。     

荧光定量PCR法检测鼻咽癌血浆游离EBV DNA的临床意义

目的通过检测血浆中游离EBV DNA水平,探讨其在鼻咽癌诊断、鉴别诊断、临床分期中的临床意义,方法采用荧光定量PCR方法定量检测111例初治鼻咽癌患者和107例放疗后持续缓解患者血浆游离EBC DNA水平．结果初治组及临床缓解组血浆中游离EBV DNA检出率分别为74．8％（83／111）和4．6％（5／107）．初治组检出率显著高于临床缓解组．在统计学上有显著性差异;初治组中晚期（Ⅲ＋Ⅳ期）血浆游离EBV DNA拷贝数显著高于早期（Ⅰ＋Ⅱ期）;N晚期（N2＋N3）拷贝数显著高于早期（N0＋N1）;T晚期（T3＋T4）拷贝数显著高于早期（T1＋T2）;统计学上均有显著性差异。结论荧光定量PCR方法检测血浆中游离EBV DNA是一种敏感可靠的方法,血浆游离EBV DNA定量检测对鼻咽癌诊断、鉴别诊断、分期、治疗后监测有重要的临床意义．有望成为鼻咽癌肿瘤学标记物。     

Hypoxia-targeting by tirapazamine (TPZ) induces preferential growth inhibition of nasopharyngeal carcinoma cells with Chk1/2 activation

Hypoxia is commonly developed in solid tumors, which contributes to metastasis as well as radio- and chemo-resistance. Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic head and neck cancer prevalent in Southeast Asia with a high incidence rate of 15–30/100,000 persons/year (comparable to that of pancreatic cancer in the US). Previous clinical studies in NPC showed that hypoxia is detected in almost 100% of primary tumors and overexpression of hypoxia markers correlated with poor clinical outcome. Tirapazamine (TPZ) is a synthetic hypoxia-activated prodrug, which preferentially forms cytotoxic and DNA-damaging free radicals under hypoxia, thus selectively eradicate hypoxic cells. Here, we hypothesized that specific hypoxia-targeting by this clinical trial agent may be therapeutic for NPC. Our findings demonstrated that under hypoxia, TPZ was able to induce preferential growth inhibition of NPC cells, which was associated with marked cell cycle arrest at S-phase and PARP cleavage (a hallmark of apoptosis). Examination of S-phase checkpoint regulators revealed that Chk1 and Chk2 were selectively activated by TPZ in NPC cells under hypoxia. Hypoxia-selectivity of TPZ was also demonstrated by preferential downregulation of several important hypoxia-induced markers (HIF-1α, CA IX and VEGF) under hypoxia. Furthermore, we demonstrated that TPZ was equally effective and hypoxia-selective even in the presence of the EBV oncoprotein, LMP1 or the EBV genome. In summary, encouraging results from this proof-of-concept study implicate the therapeutic potential of hypoxia-targeting approaches for the treatment of NPC.     

Inhibition of Epstein Barr Virus LMP1 gene expression in B lymphocytes by antisense oligonucleotides: uptake and efficacy of lipid-based and receptor-mediated delivery systems

Epstein Barr Virus (EBV), is associated with an increasing number of lymphoid and epithelial malignancies. Among the genes expressed by EBV during latency, LMP1 plays a key role for growth transformation and immortalization of B lymphocytes. We have previously shown that antisense oligonucleotides (ONs) directed to LMP1 mRNA, effectively suppressed LMP1 gene expression and substantially reduced proliferation of the infected cells. The use of antisense phosphodiester oligonucleotides as therapeutic agents is limited by inefficient cellular uptake and intracellular transport to the target mRNA. We tested the ability of three cationic carriers internalized by different pathways, to increase the delivery of anti-LMP1-ON to their site of action in EBV-infected B lymphocytes. We report here that liposomes, dendrimers or transferrin-polylysine-conjugated ON were internalized by the cells at an extent several fold higher than that of the naked oligomers. However, only the delivery system exploiting the transferrin receptor pathway of internalization, was able to vectorize biologically active antisense LMP1-ON.     

鼻咽癌放疗过程中血浆EBV DNA水平的动力学研究

目的探讨过程中鼻咽癌患者外周血EBVDNA水平的动态变化规律及与临床疗效的关系。方法运用荧光定量PCR技术动态检测22例鼻咽癌患者放疗期间血浆EBVDNA水平，放疗期间每周第1d检测1次；临床观察放疗过程中鼻咽部肿瘤消退情况与外周血EBVDNA水平变化的关系。结果放疗完成2周时。血浆EBVDNA阳性率及拷贝数较前显著下降，其余各周之间阳性率及拷贝数均无明显差异。放疗过程中血浆EBVDNA水平变化表现为三种动力学曲线形式。每周临床观察鼻咽肿块消退情况发现：随着放疗剂量增加。鼻咽肿块逐渐缩小，血浆EBVDNA水平逐渐下降。结论放疗过程中血浆EBVDNA水平变化与鼻咽癌患者临床疗效关系密切。值得进一步研究。     

The extract of Chrysanthemum indicum Linne inhibits EBV LMP1-induced NF-κB activation and the viability of EBV-transformed lymphoblastoid cell lines

Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-κB activation is essential for LCL survival. To identify a novel inhibitor candidate for LMP1-induced NF-κB activation, crude ethanol extracts of medicinal plants were screened for the potential NF-κB inhibitory activity. Seventy percent ethanol extract of Chrysanthemum indicum Linne extract (CIE) strongly reduced LMP1-induced NF-κB activation. In addition, CIE inhibited LMP1-induced IKKα or IKKβ activation. Interestingly, CIE treatment rapidly reduced LCL viability without exhibiting any adverse effects on the viability of human foreskin fibroblasts (HFF), EBV negative Burkitt’s lymphoma cell lines (BL41) or HeLa cells.     

在EBV阳性鼻咽癌细胞内SAHA联合GCV细胞毒作用的时间点分析

目的探讨伏立诺他（SAHA）联合GCV对EBV阳性鼻咽癌细胞的细胞毒作用有效时间点,为临床化疗提供依据。方法。利用Real-timePCR检测SAHA作用下EBV阳性鼻咽癌细胞C666TK的表达情况;通过MTI＂检测SAHA联合GCV的细胞毒性。结果SA—HA作用16h时TK的表达最多,并且MTr结果显示SAHA作用16h后联合GCV两者的细胞毒作用最强。结论SAHA作用16h后联合GCV抗鼻咽癌效果较好。     

Inhibition of host kinase activity altered by the LMP2A signalosome-a therapeutic target for Epstein-Barr virus latency and associated disease

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection in the majority of the human population. The virus resides in a latent state in B lymphocytes and is associated with a variety of cancers in the human host. In normal individuals, latent infection with EBV typically poses no health risk, but upon immunosuppression, either following organ transplantation or HIV infection, malignancies and lymphoproliferative diseases can result. Latent membrane protein 2A (LMP2A) is a virally encoded membrane protein that is expressed in EBV latent infection and in most of the tumors associated with EBV infection. Previous studies have indicated that LMP2A expression alters the activity of the Src family protein tyrosine kinases, the Syk protein tyrosine kinase, the Btk protein tyrosine kinase, and phosphatidylinositol 3-kinase (PI3-kinase). In this study, inhibitors of each of these kinases were tested using an in vitro system dependent on LMP2A expression for B cell colony formation in IL-7 containing methylcellulose media. Of the inhibitors tested, only piceatannol, a Syk tyrosine kinase inhibitor, demonstrated a specific effect on LMP2A expressing cells and not control cells. These studies provide a basis for targeting LMP2A function in EBV latency and may allow for the identification of novel therapeutics for the treatment or eradication of EBV latent infections and associated proliferative disorders.     

冻融人外周血树突状细胞基因疫苗体外抗肿瘤活性研究

目的观察EB病毒潜伏膜蛋白重组腺病毒(rAd-EBV-LMP2)转染的冻融人外周血树突状细胞(DC)诱导的特异性细胞毒性T淋巴细胞(CTL)抗肿瘤活性。方法人外周血来源的单核细胞经细胞因子扩增培养为成熟DC,液氮冻存;rAd-LMP2重组腺病毒转染冻融DC制备疫苗。动态观察细胞形态学特征,四甲基偶氮唑蓝(MTT)法检测DC疫苗刺激同种T淋巴细胞增殖活性,以及诱导的CTL对肿瘤细胞的杀伤活性。结果rAd-LMP2转染的冻融DC疫苗具有形态迥异、多突起的典型形态学特征,其刺激同种T淋巴细胞的增殖能力明显高于对照组(P<0.01),能诱导高效的CTL活性(P<0.01),与新鲜DC疫苗差异无统计学意义(P>0.05)。结论rAd-LMP2转染冻融DC疫苗在体外能激发较强的EBV-LMP2特异性的功能性CTL,为EBV相关的鼻咽癌(NPC)的免疫治疗提供策略。