鼻咽癌癌前病变细胞学标准的探讨

一、病理组织学基础 从人类及实验性肿瘤研究表明,鼻咽粘膜上皮的癌变,必经增生阶段,特别是异型增生(Atypical Hyperplasia),鼻咽部的早期癌(原位癌、早期微小浸润癌)是由异型增生及异型化生(Atypical metaplasia)转变而来。异型增生、异型化生的细胞即相当于涂片中的核异质(Dyskaryosis)细胞。单纯增生和单纯化生与癌变无直接关系。 从大量鼻咽癌癌旁组织的观察及大面积普查所发现的有切片对照的早期鼻咽癌来看,鼻咽原位癌多由异型增生、异型化生的细胞逐步     

基因转移与鼻咽细胞癌变的研究 Ⅱ.EB病毒基因插入宿主细胞的表达和基因产物

EB病毒引起鼻咽细胞癌变(始动因子)抑或对巴癌变的细胞起促进作用(促进因子),至今尚未有定论。不过癌前期增生细胞甚至正常鼻咽上皮细胞检出可能与细胞转化有关的EB病毒基因产物(EBNA)和EB病毒核酸的存在,是EB病毒在鼻咽癌变中起重要作用的线索。对于这种外加基因的进入方式,我们提出过不需通过病毒颗粒直接感染或细胞融合传递,可以通过DNA插入的基因转移方式进入宿主细胞。本文着重报导采用超声超滤方法和核酸原位杂交技术,分析EB病毒DNA的大分子和     

鼻咽癌前期病变中的EB病毒感染

背景与目的：鼻咽癌中的浸润性癌细胞均感染了EB病毒（Epstein-Barr virus．EBV）。前期病变可见于早期鼻咽癌癌旁上皮。本研究旨在通过检测前期病变中的EB病毒,探讨EB病毒感染存鼻咽癌变过程中的作用,及其基因型在鼻咽癌变过程中发生的宿主内演变。方法：采用核酸原位杂交检测15例早期鼻咽癌活检组织中的EB病毒编码RNA（EBV—encoded RNA,EBER）。采用巢式PCR法检测前期病变和癌巢中的EB病毒类型和潜伏膜蛋白1（latent membrane protein 1,LMPI）EB病毒株。具有代表性的LMPI基因羧基末端PCR产物采用四色荧光终止序列技术进行DNA序列分析。结果：所有15例早期鼻咽癌中的绝大多数浸润性癌细胞均呈EBER阳性。在15例的期病变中．14例可检测到EBER阳性的异常上皮细胞和／或浸润性淋巴细胞。单个A型EB病毒可在9例癌巢（11例适用）及9例前期病变（10例通用）的DNA样本中检测到。EB病毒LMP1基因羧基末端在15例癌巢DNA样本中均可检测到,其中14例是30bp缺失型LMP1 EB病毒株,1例是野生型和30bp缺失型LMP1株的混合感染。在11例适合做EB病毒LMP1基因羧基末端扩增的前期病变的DNA样本中,5例呈野生型和30bp缺失型LMP1 EB病毒株的混合感染,4例是单个缺大型LMP1 EB病毒株感染,1例呈单个野生型LMP1 EB病毒株感染,1例呈阴性反应。野生型LMP1基因羧基末端的DNA序列与B95—8细胞的DNA序列完全一致;30bp缺大型LMP1基因羧基末端的DNA序列却其有30bp缺失（密码子：346～355）和4个错义点突变（密码子：334、335、338和366）。结论：鼻咽上皮细胞的EB病毒感染是癌变过程中侵袭前的事件;而在鼻咽癌变过程中,EB病毒基因型会产生宿主内的演变。     

EB病毒DNA在鼻咽病变中的表现

本文通过原位杂交技术检测鼻咽活检组织不同病理改变时EBV—DNA的表现,以探讨EB病毒在鼻咽癌发生过程中的可能作用。结果显示:(1)本实验所有鼻咽低分化癌癌组织均出现EBV—DNA片段,且绝大部分病例的阳性细胞数及细胞阳性强度均在中等以上;(2)无论是鼻咽癌或慢性鼻咽炎病例,所见到的中-重度异型改变上皮或淋巴组织,均有数量不等的细胞存在阳性强度不同的EBV-DNA片段,粘膜下小涎腺内亦常见到一定数量的EBV-DNA片段。结果提示:(1)EB病毒与鼻咽低分化癌关系密切。EBV作为致鼻咽癌病因多因素中的一员是有可能的;(2)患者血清中EB病毒相关抗体,尤其是IgA抗体阳性率和滴度的升高,可能与鼻咽局部EBV感染及/或EBV激活有关;(3)在异型改变上皮中有中等或以上阳性强度EBV-DNA片段的检出,将有可能作为推测鼻咽癌癌前病变的有效指标。     

鼻咽癌患者外周血及肿瘤原发部位的病毒检测及意义

鼻咽癌是我国常见的恶性肿瘤,EB病毒（Epstein-Barr virus,EBV）感染是鼻咽癌发生的重要因素.鼻咽上皮细胞的EBV感染是癌变过程中侵袭前的变化,几乎在100％的鼻咽癌活检组织中存在EBV的基因.目前,利用聚合酶链反应（polymerase chain reaction,PCR）技术检测鼻咽癌患者血浆中EBV-DNA已应用于临床,检出率达96％,而传统的原位杂交方法在鼻咽癌组织中EBV编码的小RNA（EBV-encoded small RNA,EBER）仍有显著表达.本文在鼻咽癌患者外周血及鼻咽部活检组织石蜡标本中联合检测鼻咽癌患者外周血中EBV-DNA载量与鼻咽癌组织中EBER表达的差异,探讨EBV感染在鼻咽癌变过程中的变化,并对同一患者的标本进行两种方法的研究.     

鼻咽癌癌前及其高危人群鼻咽上皮组织EB病毒DNA酶基因检测

目的探讨非癌鼻咽上皮细胞发展为鼻咽癌（ＮＰＣ）的过程中,ＥＢ病毒在什么阶段进入鼻咽上皮。方法利用广州中山医科大学肿瘤防治中心四会县防癌基地１９９４年度复查工作及肿瘤医院门诊,收集鼻咽癌及各类“癌前状态”及“癌前病变”患者蜡块４５０余例,应用改进后的多聚酶链反应（ＰＣＲ）方法进行ＥＢＶＤＮＡ酶基因片段扩增检测。结果ＮＰＣ浸润癌组织ＥＢＶＤＮＡ酶基因片段扩增阳性率为９７．３％（１４５／１４９）,ＮＰＣ原位癌４例中,基因阳性者２例,而防癌基地“癌前状态”人群１５５例中阳性者２例,阳性率为１．３％（２／１５５）。临床癌前病变组阳性率为零（０／４７）。结论ＥＢ病毒在非癌鼻咽组织中出现频率十分低,ＥＢ病毒可能在鼻咽上皮癌变后才进入鼻咽上皮,提示ＥＢ病毒可能不是ＮＰＣ的病因。     

表皮生长因子受体在宫颈癌及宫颈癌前病变组织中的表达

表皮生长因子受体（ＥＧＦ－Ｒ）是原癌基因。ｃ－ｅｒｂＢ－１（也称ＨＥＲ－１）的表达产物,它与肿瘤形成的关系日渐受到人们的重视。本研究采用免疫组化方法检测正常宫颈粘膜。宫颈上皮异型增生及宫颈鳞癌组织中ＥＧＦ－Ｒ的表达,探讨ＥＧＦ－Ｒ与宫颈癌发生及生物学行为的关系。１材料与方法１．１材料收集宫颈活检及子宫切除标本１３６例,其中正常宫颈粘膜１０例,宫颈上皮轻度异型增生２２例,中度异型增生１８例,重度异型增生１５例,原位癌８例,早期浸润癌（癌组织浸润间质深度为距基底膜５ｍｍ之内）１５例,浸润癌（癌组织浸…     

应用原位核酸分子杂交法对TPA促进EB病毒基因插入宿主细胞DNA的观察

应用DNA介导的基因转移法和原位的核酸分子杂交技术,本文研究了促癌因子与EBV基因对鼻咽上皮性细胞的协同作用。(1)研究结果再次证明EB病毒基因片段可不需经完整病毒颗粒的感染而传递,也不一定经过细胞融合状态下EBV基因由淋巴细胞输入上皮细胞内,而是通过基因直接掺合和插入的方式实现EBV基因转移的生物学作用。(2)TPA有促进EBV基因插入的生物学效能。(3)TPA还能促进插入的EBV基因表达,后者反映在胞质中存在有大量mRNA和EBV基因编码的基因产物-EBNA的出现。 本文提供了一个研究EBV转化基因与促癌因于协同作用的实验模型,为今后癌变机制研究打下重要基础。     

鼻咽癌研究进展

1鼻咽癌病因学的研究1．1EB病毒自从国外学者首先报告EB病毒与鼻咽癌发病可能有关后,我国学者证实了鼻咽癌组织中有EBVDNA和EB病毒基因产物的表达,而具有转化活性的EB病毒潜伏膜蛋白1在不同分化程度的鼻咽癌组织中其基因结构与功能存在一定的差异。同时建立了携带EB病毒的鼻咽癌细胞系SUNE－1和裸鼠移植瘤株SUNT－1。近年来有研究显示：人胚鼻咽粘膜在EBV、TPA和正丁酸盐的协同作用下可诱发淋巴瘤和未分化癌。但EBV在人鼻咽癌发生发展中的确切作用机理仍未彻底阐明。1．2遗传易感性我国自70年代的死亡回顾调查、肿瘤登记、…     

鼻咽癌组织学类型与EB病毒感染的关系

[目的]研究不同组织学类型鼻咽癌与EB病毒感染的关系.[方法]收集4种主要组织学类型的鼻咽癌288例,用原位杂交方法检测癌巢及肿瘤浸润淋巴细胞的EB病毒编码小RNAs(EBERs)的表达,其中EBERs阳性的31例非角化性癌和19例角化性鳞状细胞癌,进一步用原位杂交法检测EB病毒溶解期产物EA-D(early antigen-diffuse,EA-D)mRNA的表达.[结果]接近100%的鼻咽非角化性癌(99.32%,145/146)显示出EBERs阳性信号,鼻咽腺癌EBERs阳性率明显小于角化性鳞状细胞癌,分别是35.90%(14/39)、84.38%(81/96).双向分化的腺鳞癌的EBERs阳性率(71.43%,5/7)处于非角化性癌/角化性鳞状细胞癌(93.39%,226/242)与腺癌(35.90%,14/39)之间.非角化性鳞状细胞癌、腺癌、角化性鳞状细胞癌分别有23例(23/146,15.75%)、16例(41.03%,16/39)、31例(31/96,32.29%)可见表达EBERs的肿瘤浸润淋巴细胞.角化性鳞状细胞癌的EA-D mRNA表达率高于非角化性癌,分别是78.95%(15/19)、16.13%(5/31).[结论]4种不同组织学类型鼻咽癌的EB病毒感染率与感染状态不完全一致.鼻咽非角化性癌总是与EB病毒的潜伏感染密切相关,角化性鳞状细胞癌组织中的分化不良成分也与EB病毒潜伏感染关系密切,其中分化良好的癌细胞经常可以检测到EB病毒的溶解性感染产物表达.鼻咽腺癌与EB病毒感染的关系并不密切.     

A propopsal concerning the histological typing of primary nasopharyngeal carcinoma

Primary nasopharyngeal carcinoma (NPC) denotes a carcinoma originating from the surface and crypt lining epithelium as well as the epithelial cells of minor salivary gland in the nasopharynx. It is well known that the histologic typing of primary NPC was most controversial and confusing in the past literature.[1,2] Many classifications were presented; but for consistency's sake, the classification proposed by the World Health Organization (WHO) in 1991[3] is recommended nowadays. On anoth…     

The oncogenic potential of Epstein-Barr virus.

Epstein-Barr (EB) virus is a herpesvirus which is the causative agent of infectious mononucleosis. It can also be classified as a human DNA tumor virus as it is also etiologically associated with the development of Burkitt's lymphoma (BL) and nasopharyngeal carcinoma. EB virus involvement in these human tumors is suggested by seroepidemiological studies and the detection of virus DNA and proteins in tumor biopsies. Furthermore, EB virus can be detected in lymphoproliferations and malignant tumors arising from immunosuppressed individuals which further emphasizes its oncogenic potential. EB virus immortalizes B lymphocytes in vitro giving rise to continuously proliferating lymphoblastoid cell lines (LCL). These LCL are not tumorigenic, which suggests that cellular infection by virus alone is not sufficient for full tumorigenicity. This has led to the development of a multistep scenario for the development of BL where EB virus, in conjunction with other suggested cofactors, is necessary to induce the tumorigenic phenotype.     

Activation in vitro by BUdR of a productive EB virus infection in the epithelial cells of nasopharyngeal carcinoma.

Material from a nasopharyngeal carcinoma (NCP) has been passaged in athymic (nude) mice to eliminate non-malignant infiltrating cells. The human origin and derivation from NPC malignant epithelial cells of the nude mouse tumours have been confirmed by chromosome examination, electron microscopy showing desmosomes and keratin fibrils, and postive EB virus nuclear antigen (EBNA) testing. Samples of the mouse-grown tumours were cultured and pure monolayers of epithelial cells were obtained which still expressed EBNA and contained desmosomes and keratin; these cultures grew well for about 3 weeks. Extensive electron microscope searches failed to reveal herpes virus particles. In contrast, cultures treated with BUdR showed typical immature and mature herpes virus particles in epithelial, keratin-containing cells, and immunofluorescence tests for virus capsid antigen with a battery of human sera identified this agent as EB virus. EB virus has thus, for the first time, been activated in NPC epithelial cells and shown to be capable of replication in a cell type other than a primate B-lymphocyte.     

应用FQ-PCR技术测定鼻咽癌患者血浆EB病毒DNA定量分析及其临床意义

目的 应用FQ-PCR技术测定鼻咽癌患者血浆中游离EB病毒DNA拷贝,探讨血浆EB病毒DNA定量测定的临床意义.方法 选取132例鼻咽癌患者,取其外周血样本,其中84例治疗前血样,48例治疗后血样(放疗或加化疗).另收集60例健康血样作为正常对照.使用广州中山医科大学达安基因诊断中心提供(Cat.#DA-061)的EB病毒DNA-PCR试剂盒,测定鼻咽癌患者血浆中游离EB病毒DNA拷贝,阴性对照为空白PCR反应液,阳性对照为106、104、102拷贝/ul的阳性模板.结果 鼻咽癌组治疗前84例样本,阳性率为67.86%,鼻咽癌组治疗后48例样本阳性率为35.42%,正常对照者30例样本阳性率仅为8.33%,鼻咽癌组治疗前血浆游离EB病毒DNA的阳性率显著高于对照组,差异显著(χ2=11.497,P=0.001);鼻咽癌组治疗后与对照组血浆游离EB病毒DNA的阳性率比较,差异较显著(χ2=6.782,P=0.018);鼻咽癌组治疗前血浆中游离EB病毒-DNA阳性率约是治疗后的2倍,差异显著(χ2=6.271,P=0.023).鼻咽癌组治疗前血浆游离EB病毒DNA拷贝中位数为522.0 copies/ml,治疗后中位数为0.0,对照组中位数为0.0,鼻咽癌组治疗前的血浆游离EB病毒DNA拷贝数显著高于治疗后,两者差异具有统计学意义(U=350.0,P=0.029),而且与正常对照组拷贝数比较,差异亦显著(U=274.0,P=0.001).Ⅰ~Ⅱ期患者的血浆游离EB病毒DNA水平显著低于Ⅲ~Ⅳ期患者(U=141.0,P=0.039).N0+N1期患者的血浆EB病毒DNA水平显著低于N2+N3期患者(U=147.0,P=0.031).结论 FQ-PCR技术具有快速、精确和高灵敏性的特点,比其它传统检测手段更实用.血浆EB病毒DNA的定量PCR分析对鼻咽癌的筛选检查具有应用价值.     

Expression of Etk/Bmx tyrosine kinase in the tumorigenicity of nasopharyngeal epithelium and its relation with EB virus infection and the apoptosis-related protein Bcl-2

We compared Etk/Bmx expression in nasopharyngeal carcinoma (NPC) and non-neoplastic nasopharyngeal lesions in order to learn whether the expression of this non-receptor protein tyrosine kinase is associated with the development of NPC. We also related Etk/Bmx expression to factors resulting from Epstein-Barr virus (EBV) infection. We used immunohistochemistry (IHC) and in situ hybridization to examine 20 non-neoplastic nasopharyngeal lesions and 49 cases of NPC to assess Etk/Bmx, EB virus latent membrane protein-1 (LMP-1), Bcl-2 and EBV-encoded small RNA-1 expression in these samples. Etk/Bmx expression was present in the basal cell nuclei of the nasopharyngeal epithelium in 1/9 (11.1%) cases of chronic nasopharyngitis and 2/11 cases (18.2%) of dysplasia. While 13/49 (26.5%) NPC cases expressed Etk/Bmx, the difference in frequency between the expression of Etk/Bmx in the non-neoplastic and NPC cases was not significant. Etk/Bmx expression was correlated with the presence of EBER-1 immunopositivity in dysplasia and in NPC but not in chronic nasopharyngitis. The presence of Etk/Bmx immunopositivity was independent of the expression of either LMP-1 or Bcl-2 in either the nasopharyngeal carcinoma or the non-neoplastic lesions. This suggests that in some cases of non-neoplastic and neoplastic nasopharyngeal lesions, Etk/Bmx may participate in regulating epithelial differentiation. While EBV-related small RNA-1 may participate in this regulation, neither LMP-1 or Bcl-2 expression appears to be related to Etk/Bmx expression.     

Isolation of infectious EB virus from the epithelial tumour cells of nasopharyngeal carcinoma.

Evidence of herpesvirus replication has been found by light and electron microscopy in the malignant epithelial cells of two out of six nasopharyngeal carcinomas (NPC) examined directly after growth in nude mice to eliminate non-malignant infiltrating cells. The agent has been identified as EB virus by immunofluorescence tests for EB virus capsid antigen, and has been shown to be biologically active by its ability to infect and transform foetal cord blood lymphocytes. Lymphoblastoid cell lines which express the EB virus nuclear antigen have been established from the transformed foetal lymphocytes, and thus carry the first isolate of the virus from the actual epithelial tumour cells of NPC, in a form suitable for further investigation. The results are discussed in terms of the relationship of EB virus to NPC epithelial cells.     

鼻咽癌中EB病毒LMP1基因N端XhoⅠ酶切位点的丢失

背景与目的:众所周知, EB病毒 LMP1基因在鼻咽癌变过程起着一定的作用.本研究通过检测广东地区鼻咽癌组织 EB病毒 LMP1基因 N-末端区 XhoⅠ酶切位点的丢失,探讨 LMP1基因变异在鼻咽癌发生发展中的作用.方法 :收集中山大学肿瘤防治中心鼻咽癌患者鼻咽新鲜活检标本 63例.收集 EB病毒健康携带者外周血单个核细胞 (PBMCs) 10例作为对照.采用 QIAamp DNA Mini Kit和 QIAamp DNA Blood Mini Kit分别抽取组织和外周血单个核细胞的 DNA,应用巢式 PCR扩增 EB病毒 LMP1基因的 N-末端区,并用 XhoⅠ对扩增产物进行酶切.采用四色荧光 末端终止法对扩增产物进行序列分析.结果: 10例健康携带者外周血单个核细胞的 EB病毒 LMP1基因 N-末端区均未见 XhoⅠ酶切位点的丢失.63例鼻咽癌组织中有 50例(79.37%)出现 XhoⅠ酶切位点的丢失(XhoⅠ-loss),还有 4例(6.34%)为 XhoⅠ酶切位点部分丢失,只有 9例(14.29%)未见 XhoⅠ酶切位点的丢失(wt-XhoⅠ ).除了 XhoⅠ酶切位点的丢失 (nt: 169423～169428; GAGCTC→ GATCTC)外,还发现四个错义点突变.结论:本研究所检测的广东地区 EB病毒健康携带者外周血单个核细胞所携带的 EB病毒 LMP1基因为 wt-XhoⅠ,而在鼻咽癌组织中主要为 XhoⅠ-loss.因此,我们认为 EB病毒 LMP1基因 N-末端区 XhoⅠ酶切位点的丢失和其他的错义点突变可能是在鼻咽癌的发生发展过程中产生的.     

Cytomegalovirus isolations from cell cultures derived from Epstein-Barr virus-associated nasopharyngeal carcinoma

Cytomegalovirus (CMV) was isolated in cell cultures derived from 2 of 11 nasopharyngeal carcinoma (NPC) biopsy specimens from North African patients. All these cases were Epstein-Barr virus (EBV)-associated NPC. Morphologic cytopathic changes and viral replication not associated with EBV were observed after 2 months in culture. Virus identification was achieved by immunofluorescence studies, and cell culture antigens were tested by the use of complement fixation and indirect hemagglutination. All these NPC patients had been infected by herpes simplex virus, varicella-zoster virus, and CMV, but the antibody titers determined by complement fixation and immunofluorescence were normal. CMV, which is not associated with this cancer, could nevertheless favor carcinogenesis in facilitating fusion between epithelial cells and EBV-positive lymphocytes.