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Molecular biologic and pathologic study of Epstein-Barr virus infected lymphepithalial carcinomas in salivary gland

EB病毒感染唾液腺淋巴上皮癌的分子生物学和病理学研究

基本信息

批准号：
12576024
负责人：
SAKU Takashi
金额：
$ 9.73万
依托单位：
NIIGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12576024/
关键词：
lymphoepithelial carcinoma salivary gland tumor EBV LMP1 gene gene polymorphism cell culture PCR carcinogenetic mutations DNAシークエンス LMP-1 30bp欠損 in-situハイブリダイゼーション中国ロシア

项目摘要

A total 180 cases of salivary gland lymphoepithelial carcinoma (LEC) were col lected from Russia-Asian countries. Their paraffin blocks of surgical materials were obtained for imunohistochemistry, in-situ hybridization and DNA extractionImmunohistochemically, about 50% of the cases showed positive signals for LMP1 gene products, and 100% of them had EBER-1 RNA signals in tumor cells' nuclei, which suggested a definite EBV infection among salivary LEC cases. From 40 cases of them, the C-terminus of the LMP1 gene was PCR-amplified and direct-sequenced. The results indicated that there were at least two different strains of EBV harboring in salivary LEC and that there were many mutational events in them including the characteristic 30 by delefion. From 12 cases of them, the complete sequence for, the LMP1 gene were obtained, and many and new mutations in addition to those which were already found in nasapharyngeal LEC. The promoter region of the LMP1 gene was also analyzed from 20 cases o … More f them, and all of the cases shared common mutational events in regions which were expected to be associated with transcription factorsOne of the complete sequence obtained as mentioned above were cloned into an expression vector and the constructs were transfected into 293T cells to determine their effectd on the activation of NFkB pathways as well as cell growth. The transfected cells showed significant elevation of NFkB activities and suppressed cell growth, which were also seen in cells transfected with vectors with nasopharyngeal CAO or prototype B95-8. The results indicated that the expression of LMP1 affects transcritption activities in LEC cells but that the overall mutaions found in the present study were not always reasons for the tumorigenesisPrimary cultures for salivary LECs were tried many times. However, the cells were hard to maintain. One of them are growing now and they were characterized by immunofluorescence and PCR to be epithelial and EBV infectedThe results obtained as above has given us a new insight for EBV roles in the pathogenesis of salivary LEC. To this end, it is necessary to carry out further investigations on molecular functions of EBV oncogenes Less

共收集了180例来自俄罗斯和亚洲国家的唾液腺淋巴上皮癌（LEC）病例。获得手术材料石蜡块进行免疫组化、原位杂交和DNA提取。免疫组化结果显示，约50%的病例显示LMP1基因产物阳性信号，其中100%的肿瘤细胞核内有EBER-1 RNA信号，这表明唾液LEC病例中有明确的EBV感染。对其中40例的LMP1基因C末端进行PCR扩增并直接测序。结果表明，唾液LEC中至少存在两种不同的EBV毒株，其中存在多种突变事件，其中特征性缺失的突变事件有30个。从其中的12例中，获得了LMP1基因的完整序列，以及除了在鼻咽LEC中已经发现的突变之外，还获得了许多新的突变。还对 20 个病例的 LMP1 基因启动子区域进行了分析，所有病例在预计与转录因子相关的区域中都有共同的突变事件。将上述获得的完整序列克隆到表达载体中，并将构建体转染到 293T 细胞中，以确定它们对 NFkB 通路激活以及细胞生长的影响。转染的细胞表现出 NFkB 活性显着升高并抑制细胞生长，这在用鼻咽 CAO 或原型 B95-8 载体转染的细胞中也可见。结果表明，LMP1的表达影响LEC细胞的转录活性，但本研究中发现的整体突变并不总是肿瘤发生的原因。唾液LEC的原代培养已进行了多次尝试。然而，这些细胞很难维持。其中一个正在生长，通过免疫荧光和PCR鉴定为上皮和EBV感染。上述结果使我们对EBV在唾液LEC发病机制中的作用有了新的认识。为此，有必要对EBV癌基因的分子功能进行进一步研究。