良、恶性鼻咽活检组织中EB病毒DNA检测、EBNA的分型及表达

应用ＥＢ病毒ＤＮＡＢａｍＨＩＷ片段、ＥＢＮＡ一２Ａ、Ｂ型分了探针，检测我区鼻咽癌（包括鼻咽原位癌）、临床高度怀疑鼻咽癌（包括鼻咽上皮细胞非典型增生、颈淋巴结肿大及鼻咽结节）和慢性鼻咽炎等活检组织共９６例。各组均以Ａ型病毒感染为主：鼻咽癌组阳性为２７／３５（７７．１％）；临床疑癌组７／１０（７０．０％）；慢性鼻咽炎织１８／２７（６６．７％）。Ｗ和Ａ型均阳性的鼻咽癌组为２０／２９（６９．０％）；非癌组为８／２５（３２．０％）。同时用抗补体免疫荧光法检测ＥＢＮＡ的表达，鼻咽癌组阳性为１９／２０（９５．０％），而慢性炎组为１／１５（６．７％）。以ＥＢ病毒ＤＮＡＢａｍＨＩＷ片段、ＥＢＮＡ一２Ａ型片段和ＥＢＮＡ表达检测中任一项阳性作为ＥＢ病毒感染、整合或表达的依据，则鼻咽癌组与非癌组之间有明显的差别（Ｐ＜０．００１）。这从另一角度提示ＥＢ病毒（特别是ＥＢＮＡ一２Ａ型）感染与我区鼻咽癌病因、发病有密切关系。     

良,恶性鼻咽活检组织中EB病毒DNA检测,EBNA的分…

应用ＥＢ病毒ＤＮＡＢａｍＨＩＷ片段，ＥＢＮＡ－２Ａ，Ｂ型分子探针，检测我区鼻咽癌（包括鼻咽原位癌），临床高度怀疑鼻咽癌（包括鼻咽上皮细胞非典型增生，颈淋巴结肿大及鼻咽结节）和慢性鼻咽炎等活检组织共９６例。各组均以Ａ型病毒感染为主：鼻咽癌组织性为２７／３５（７７．１％）。临床疑癌组７／１０（７０．０％）；慢性鼻咽炎组１８／２７（６６．７％）。Ｗ和Ａ型均阳性的鼻咽癌组为２０／２９（６９．０％）；非癌组     

一个定位于染色体3p25.3的新基因及在鼻咽癌中的表达分析

目的 分离位于染色体３ｐ２４－２６区域中鼻咽癌相关的新基因。方法 在染色体３ｐ２４－２６鼻咽癌杂合性丢失（ｌｏｓｓ ｏｆ ｈｅｔｅｒｐｚｙｇｏｓｉｔｙ,ＬＯＨ）高频区,用ＲＴ－ＰＣＲ及Ｎｏｒｔｈｅｒｎ杂交,检测了２０个表达序列标记（ｅｘｐｒｅｓｓｅｄ ｓｅｑｕｅｎｃｅ ｔａｇ,ＥＳＴ）在鼻吕细胞株ＨＮＥ１和原代培养的正常鼻咽上皮细胞中的表达水平,并对其中一个在鼻咽癌细胞株ＨＮＥ１中表达下调的ＥＳＴ     

鼻咽癌组织中人疱疹病毒6型感染及其临床意义

目的　探讨人疱疹病毒６ 型（ＨＨＶ６） 与鼻咽癌（ＮＰＣ） 发病的关系。方法　采用聚合酶链反应（ＰＣＲ） 和原位杂交同时检测正常鼻咽组织和ＮＰＣ 癌前鼻咽组织以及ＮＰＣ 组织中ＨＨＶ６ 和ＥＢＶＤＮＡ 的存在情况。采用免疫组化检测３６ 例ＮＰＣ 组织的ＥＢＶＬＭＰ１ 蛋白表达。结果　在４２ 例ＮＰＣ组织、２１ 例鼻咽癌前病变组织中,经ＰＣＲ检出ＨＨＶ６ ＤＮＡ,阳性率分别为３０－９％ （１３ 例） 和４－７ ％（１ 例） 。ＥＢＶＤＮＡ为９５－２％ （４０ 例） 和６６－６ ％ （１４ 例）。２７ 例正常鼻咽组织未检出ＨＨＶ６ ＤＮＡ,ＥＢＶＤＮＡ为２５－９％ （７ 例）。经ＩＳＨ,仅在１３ 例ＰＣＲＨＨＶ６ ＤＮＡ阳性的ＮＰＣ组织中检出１１ 例阳性,３６ 例ＮＰＣ组织、１０ 例鼻咽癌前病变组织ＩＳＨＥＢＶＤＮＡ 阳性。ＮＰＣ组织的ＥＢＶＬＭＰ１ 蛋白阳性率为４７－２ ％（１７３６）。结论　鼻咽癌组织中存在ＨＨＶ６ 感染,提示ＨＨＶ６ 感染与ＮＰＣ有关。ＨＨＶ６ 感染与ＥＢ病毒ＬＭＰ１ 蛋白表达上调呈正相关,提示在ＮＰＣ 的发生中ＨＨＶ６ 可能直接参与和（ 或） 通过上调ＥＢＶＬＭＰ１ 的表达而起一定作用     

EB病毒感染对鼻咽上皮HLA表型改变的影响

用碱性磷酸酶抗碱性磷酸酶法（ＡＰＡＡＰ）检测ＨＬＡ－Ⅰ和ＨＬＡ－Ⅱ的表达。结果显示，体外培养的１０例人胚鼻咽上皮细胞被ＥＢ病毒感染后，ＨＬＡ－Ⅰ型抗原的表达无明显改变（阳性细胞为８５．８％±１７．１６％）；但ＨＬＡ－Ⅱ抗原的表达比对照组明显增高，两组阳性细胞各为５２．４％±１７．１６％和９．６７％±７．２３％，其差别有显著的统计学意义（Ｐ＜０．０５）。鼻咽活检组织中，１８例鼻咽癌细胞同时表达ＨＬＡ－Ⅰ和－Ⅱ两种抗原，并以后者为主（各为７２．２％和１００％），聚合酶链反应（ＰＣＲ）查到ＥＢ病毒ＤＮＡ的占９０％，与慢性鼻咽炎的差别有显著的统计学意义（Ｐ＜０．０５）。ＨＬＡ－Ⅱ表达增高可能在鼻咽癌的发生和发展中起着一定的作用。     

鼻咽癌组织基因表达谱的研究

目的和方法：用Ｃｌｏｎｔｅｃｈ公司的Ａｔｌａｓ＾ＴＭＨｕｍａｎ Ｃａｎｃｅｒ ｃＤＮＡ Ｅｘｐｒｅｓｓｉｏｎ Ａｒｒａｙ对人胚鼻咽、正常成人鼻咽、ＨＮＥ１细胞、鼻咽低分化鳞癌、肺癌和头颈部其它３种肿瘤５８８个与肿瘤相关基因的表达谱进行了研究。结果：在选定的９１个基因中,有３６个基因的鼻咽癌组织中表达加强,５１个基因鼻咽癌组织中表达下调,在鼻咽癌组织中表达下调的基因相对较多。周期素依赖性刺酶抑制子－     

八株鼻咽癌细胞株的Rb基因及其表达

为观察８株鼻咽癌细胞株（ＣＮＥ１、ＣＮＥ２、ＨＮＥ１、ＨＮＥ２、ＨＮＥ３、ＨＯＮＥ１、ＳＵＮＥ１和ＨＫ１）Ｒｂ基因的结构和表达情况。在Ｓｏｕｔｈｅｒｎ杂交，除ＨＫ１出现一微弱的额外条带外，其余７株未见异常。聚合酶链反应分析发现ＣＮＥ１和ＳＵＮＥ１的Ｒｂ基因外显子１０和ＨＯＮＥ１的外显子１３扩增阴性。免疫沉淀／免疫印迹可见８株鼻咽癌细胞株均出现分子量正常的Ｒｂ蛋白条带。免疫组织化学则见生长活跃的细胞中强阳性反应细胞数远较静止和衰老的细胞为多。综上所述，８株鼻咽癌细胞株中４株可能有微小的Ｒｂ基因变化。免疫组化强阳性可能表示８株鼻咽癌细胞株部分细胞Ｒｂ基因表达异常。     

促癌剂协同下Epstein-Barr病毒体内外感染引起人胚鼻咽上皮的形态学改变

目的探讨在促癌剂协同下Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）对人胚鼻咽上皮的感染和转化能力。方法体外培养的人胚鼻咽上皮分别接受ＥＢＶ、ＥＢＶ＋佛波酯（ＴＰＡ）或ＴＰＡ处理，不同时间测量细胞生长晕大小，收集细胞，涂片，ＨＥ染色作形态测量；移植了人胚鼻咽组织的裸小鼠分别接受多次ＥＢＶ、ＥＢＶ＋ＴＰＡ皮下注射，处死动物后对移植物作常规病理切片观察。结果体外培养的人胚鼻咽上皮在ＥＢＶ转染后（单用ＥＢＶ或在ＴＰＡ协同下），生长能力增加，核浆比增大；在体内，ＥＢＶ感染（单用ＥＢＶ或在ＴＰＡ协同下）可引起移植于裸小鼠的人胚鼻咽组织癌变（原位癌与早期浸润癌各１例）。在体外，单独使用ＴＰＡ没有诱癌作用，但似能增加ＥＢＶ的感染率，尤其在早期；在体内，在ＴＰＡ协同下，ＥＢＶ能诱发早期浸润癌。结论提示ＥＢＶ在体外能使人胚鼻咽上皮细胞出现恶性转化倾向，及引起移植于裸小鼠的人胚鼻咽组织癌变，ＴＰＡ能促进ＥＢＶ感染和致癌作用。     

人乳头状瘤病毒18型E6E7基因诱导人胚食管上皮永生化

目的为研究病毒和肿瘤的关系,用人乳头状瘤病毒（ＨＰＶ）１８型Ｅ６Ｅ７基因感染胎儿食管上皮,建立一株新的人食管上皮永生化细胞株（ＳＨＥＥ）。方法ＨＰＶ１８Ｅ６Ｅ７腺病毒伴随病毒（ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ））载体的构建;胚胎食管组织培养,ＨＰＶ１８Ｅ６Ｅ７ＡＡＶ感染,继续培养传代。用光镜、电镜检查其形态;聚合酶链反应（ＰＣＲ）、荧光原位杂交（ＦＩＳＨ）检查该病毒片段;用软琼脂培养和裸鼠接种检查致瘤性。结果经过长时间的传代培养,ＳＨＥＥ的表型仍保留原代上皮细胞培养的特征,表现为单层生长和锚锭依赖性生长,在软琼脂培养不形成克隆,接种裸鼠未成瘤。ＳＨＥＥ细胞系电镜检查可见张力原纤维,免疫组织化学检查细胞角质蛋白阳性,证实为鳞状上皮来源。ＦＩＳＨ和ＰＣＲ检测显示有ＨＰＶ１８Ｅ６Ｅ７基因。结论用ＨＰＶ１８Ｅ６Ｅ７基因建立食管上皮永生化细胞株ＳＨＥＥ,支持ＨＰＶ１８可能和食管癌病因有关的观点,可进一步用以研究食管癌的病因和发病机制。     

促癌剂协同下Epstein—Barr病毒体内外感染引起人胚鼻 …

目的 探讨在促癌剂协同下Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒（ＥＢＶ）对人胚鼻咽上皮的感染和转化能力。方法 体外培养的人胚鼻咽上皮分接接受ＥＢＶ、ＥＢＶ＋佛波酯（ＴＰＡ）或ＴＰＡ处理，不同时间测量细胞生长晕大小，收集细胞，涂片，ＨＥ染色作形态测量；移植了人胚鼻咽组织的裸小鼠分别接受多次ＥＢＶ、ＥＢＶ＋ＴＰＡ皮下注射，处死动物后对移植物作常规病理切片观察。结果 体外培养的人胚鼻咽上皮在ＥＢＶ转染后（单用ＥＢ     

cDNA代表性差异分析法分离鼻咽癌上皮细胞株HNE1表达差异cDNA序列的初步研究

目的寻找鼻咽癌中差异性表达基因，包括与鼻咽癌发病相关的候选抑瘤基因。方法应用ｃＤＮＡ代表性差异分析法（ＲＤＡ），分离原代培养的正常人鼻咽上皮细胞与鼻咽癌细胞株ＨＮＥ１中差异表达的ｃＤＮＡ序列，Ｓｏｕｔｈｅｒｎ杂交和Ｎｏｒｔｈｅｒｎ杂交被用来分析差异性表达产物的来源，最后，将这些序列克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中，并用链终止法测序。结果在第４轮杂交及扩增反应后，获得４条差异性条带。Ｓｏｕｔｈｅｒｎ杂交及Ｎｏｒｔｈｅｒｎ杂交证明，这些差异性片段来自作为“检测”扩增子的正常人鼻咽上皮，在鼻咽癌细胞株ＨＮＥ１中不表达或表达降低。序列分析这些差异性片段的克隆，发现一些序列是与已知基因高度同源的基因，包括一些看家基因；另有一些基因则为新基因序列。结论鼻咽癌的发生是一个多基因参与的过程，所获得的差异性片段中，与之同源的一些已知基因具有抑瘤功能。     

EB病毒基因组限制片段长度多态性(RFLP)在鼻咽组织中的分布

利用PCR和限制性内切酶分析,探讨了EB病毒基因组限制片段长度多态型(RFLP)在正常鼻咽组织,鼻咽癌组织和口腔上皮脱落细胞中的分布情况。结果表明在正常鼻咽组织的EB病毒感染是一种普遍现象并且感染可能发生在癌变前;EB病毒基因组BamHF区的RFLP在鼻咽癌组织和正常的鼻咽组织之间有不同的分布;不同EB病毒RFLP变型(variant)的混合感染在正常咽组织比在癌组织中普遍,这结果表明可能在癌变发展过程中,由于部分带有EB病毒的细胞的选择生长,结果使得癌组织中的细胞以一种EB病毒变型为主;本文探讨了EB病毒的RFLP变型在鼻咽组织和口腔上皮脱落细胞分布的关系,这种关系可能为鼻咽癌高发区的癌变检测提供一种简单可靠的方法。     

Differential display and cloning of messenger RNA from human normal nasal epithelial cells versus nasopharyngeal carcinoma cell lines

Carcinogenesis is a multi-process event that has been characterized both by activation of cellular oncogenes and by loss of function of tumor suppressor genes. However, no systemic study has been performed to understand the involvement of oncogenes and tumor suppressor genes in the oncogenesis of nasopharyngeal carcinoma (NPC). Differential display was performed to identify genes specifically expressed in normal nasal epithelial cells or NPC cell line HONE-1. Using Ltk3 and T11CA as primers, a 379-bp cDNA fragment (CN3) obtained from normal nasal epithelial cells was able to show specificity by northern blot analysis. A 3.5-kb mRNA was detected in normal nasal epithelial cells but not in NPC cell line HONE-1 by using 32P-end-labeled CN3 fragment as a probe. Sequence analysis of the 379 bp cDNA fragment indicated unique sequences from nts 1 to 230. Nts 231 to 379 are Alu-like sequences. Northern blot analysis using 32p-labeled PCR product amplified from nts 36 to 222 of CN3 cDNA fragment was also able to detect the 3.5-kb mRNA in normal nasal epithelial cells but not in HONE-1 and two other NPC cell lines NPC-TWO1, NPC-TWO4.     

Detection of Epstein-Barr virus DNA in nasopharyngeal carcinoma using a non-radioactive digoxigenin-labelled probe

The presence of Epstein Barr virus (EBV) DNA in biopsies from the post-nasal space (PNS) of patients suspected of nasopharyngeal carcinoma (NPC) was detected by in situ cytohybridization with an EBV DNA probe labelled with the novel labelling compound digoxigenin. The digoxigenin probe was hybridised to cryostat sections of NPC biopsies and subsequently detected by an enzyme immunoassay procedure. It was found that in situ cytohybridization using the digoxigenin probe was much more rapid and sensitive (96 h compared to five weeks) than the current method of using 3H-labelled probe. Using the digoxigenin EBV probe, it was found that in all the eighteen NPC biopsies tested, EBV DNA was detected in malignant epithelial cells and infiltrating lymphocytes. EBV DNA was also detected in some normal epithelial cells in these NPC biopsies. EBV DNA was not detected in epithelial cells of non-malignant biopsies.     

Epstein-Barr virus DNA in nasopharyngeal biopsies

The Epstein-Barr virus (EBV) has been closely associated with the undifferentiated form of nasopharyngeal carcinoma (NPC), which is particularly common in the high risk area in southeast China. We have examined 37 nasopharyngeal biopsies from patients within this high risk area, including 31 cases of undifferentiated NPC and 6 cases of patients with nasopharyngitis, for the presence of EBV DNA. We found that 26 of 31 biopsies from NPC patients were EBV DNA positive; 3 of the 6 biopsies from patients diagnosed with nasopharyngitis were also EBV DNA positive. Southern blot analysis of the DNAs obtained from the EBV genome positive biopsies, digested with EcoRI, showed that all preparations from the NPC tumors had only one band corresponding to the EcoRI A fragment when a BamHI W fragment was used as a probe. However, one tumor had an additional band with a molecular weight larger than EcoRI A. The presence of this novel band could indicate the integration of viral DNA into host cellular DNA. DNA from the same biopsies were restricted with BamHI and PstI restriction enzymes. The data obtained from these experiments suggest that the EBV genomes in both the NPC tumor biopsies and biopsies from nasopharyngitis patients obtained from an endemic area in South China may be similar to each other and to the B95-8 EBV isolate with respect to the BamHI Y region of the EBV genome. The data also demonstrate that infection of normal nasopharyngeal epithelial cells with EBV takes place in patients with nasopharyngitis.     

Identification of a novel 12p13.3 amplicon in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer commonly occurring in southern China. To decipher the molecular basis of this cancer, we performed high‐resolution array CGH analysis on eight tumour lines and 10 primary tumours to identify the genes involved in NPC tumorigenesis. In this study, multiple regions of gain were consistently found at 1q21‐q24, 7q11‐12, 7q21‐22., 11q13, 12p13, 12q13, 19p13 and 19q13. Importantly, a 2.1 Mb region at 12p13.31 was highly amplified in a NPC xenograft, xeno‐2117. By FISH mapping, we have further delineated the amplicon to a 1.24 region flanked by RP11‐319E16 and RP11‐433J6. Copy number gains of this amplicon were confirmed in 21/41 (51%) primary tumours, while three cases (7.3%) showed high copy number amplification. Among the 13 genes within this amplicon, three candidate genes, lymphotoxin beta receptor (LTβR), tumour necrosis factor receptor superfamily memeber 1A (TNFRSF1R) and FLJ10665, were specifically over‐expressed in the NPC xenograft with 12p13.3 amplification. However, only LTβR was frequently over‐expressed in primary tumours. LTβR is a member of the TNF family of receptors, which can modulate NF‐κB signalling pathways. Over‐expression of LTβR in nasopharyngeal epithelial cells resulted in an increase of NF‐κB activity and cell proliferation. In vivo study showed that suppression of LTβR by siRNA led to growth inhibition in the NPC tumour with 12p13.3 amplification. These findings implied that LTβR is a potential NPC‐associated oncogene within the 12p13.3 amplicon and that its alteration is important in NPC tumorigenesis. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

鼻咽癌组织EB病毒膜抗原和EB病毒核酸观察

目的 观察分析EB病毒膜抗原和EB病毒核酸的存在及其在鼻咽癌诊断中的作用。方法 应用抗EB病毒膜抗原（EBV／MA）的单克隆抗体检测51例病理确诊的鼻咽癌（NPC）活检标本的冰冻切片,以Southern杂交法检查20例鼻咽癌活检组织中EB病毒核酸（EBV／DNA）。结果 其中49例有EBV／MA的表达,经细胞形态学分析首次发现鼻咽癌细胞,增生上皮细胞以及正常鼻咽部上皮细胞都有BV／MA。而12例慢性炎症病例仅1例阳性,20例低分化鼻咽癌中17例存在与细胞转化有关的K片段。证实鼻咽癌组织中不仅有EB病毒核酸的重复序列W片段,还有与细胞转化有关的K片段的序列存在。结论 进一步表明EB病毒在鼻咽癌的发展中并非“过客”,而可能是其病原重要因素之一。     

Alterations of biologic properties and gene expression in nasopharyngeal epithelial cells by the Epstein-Barr virus-encoded latent membrane protein 1

Undifferentiated nasopharyngeal carcinoma (NPC) is closely associated with EBV infection, and the EBV-encoded latent membrane protein 1 (LMP1) is frequently detected in NPC. However, little is known about the pathologic roles of LMP1 in this disease. Recently, we reported the morphologic transformation and increased expression of the LAMC2 and ITGalpha6 genes in LMP1-expressing NPC cell lines. In this study, we further examine the effects of LMP1 in an immortalized nasopharyngeal epithelial cell line called NP69. This cell line was established from primary nonmalignant nasopharyngeal epithelial cells and may represent a model of premalignant nasopharyngeal epithelial cells. LMP1 induced many phenotypic changes in NP69 cells. These include morphologic transformation, increased cell proliferation, anchorage-independent growth, resistance to serum free-induced cell death, and enhanced cell migration and invasion. In addition, expression array analysis identified 28 genes that demonstrated a more than 2-fold difference in expression of NP69 cells expressing LMP1 when compared with a vector control. Two of the up-regulated genes (VEGF and vimentin) identified have been previously reported as LMP1 targets. The majority of the identified genes are associated with cell growth, differentiation, cell shape, and invasion. The present findings support the proposed roles of LMP1 in promoting cell transformation, migration, and invasion in premalignant nasopharyngeal epithelial cells. The present study also indicates the activation of the Ras/MAPK pathway in LMP1-expressing cells, which may be involved in mediating some of the transforming effects of LMP1 observed in nasopharyngeal epithelial cells.