鼻咽癌中EB病毒BamHI“f”变异和潜伏膜蛋白1基因XhoI缺失型的分析

Analysis of Epstein-Barr virus with BamHI "f" variant and XhoI-loss of LMP1 gene in nasopharyngeal carcinoma

目的 检测鼻咽癌组织中EB病毒BamHI“f”和LMP1XhoI loss基因变异并探讨其意义。方法 采用聚合酶链反应 (PCR)、巢式PCR和限制性酶切分析检测 4 0例鼻咽癌组织中EB病毒BamHI“f”和LMP1XhoI loss基因变异。对 4 8例健康成人外周血单个核细胞进行了EB病毒LMP1XhoI loss变异的检测。对 3例具有代表性的PCR产物进行了基因序列分析。结果 4 0例鼻咽癌中EB病毒BamHI“f”变异型 30例 (75 % ) ,BamHIF型 10例 (2 5 % )。 4 0例鼻咽癌组织中 39例同时进行了EB病毒LMP1XhoI loss的检测 ,30例 (76 9% )为LMP1XhoI loss;7例 (18 0 % )为LMP1Wt XhoI;2例为LMP1Wt XhoI和XhoI loss并存。 97 4 % (38/ 39)鼻咽癌中至少出现一种类型EB病毒基因变异。仅 1例鼻咽癌为EB病毒LMP1Wt XhoI/BamHIF ,基因序列分析 (LMP1第 3外显子 )发现也有 7个碱基替换 (其中5个为错义突变和 2个为同义突变 )。 4 8例健康成人外周血单个核细胞有 10例 (2 0 8% ,10 / 4 8)成功扩增出EB病毒LMP1片段 ,10例均为LMP1Wt XhoI。结论 与B95 8细胞株中EB病毒基因比较 ,鼻咽癌组织中EB病毒几乎均存在基因变异。健康成人携带的均为EB病毒LMP1Wt XhoI,而鼻咽癌细胞中主要为LMP1XhoI loss。因而 。

Objective To investigate the genomic variation of Epstein-Barr virus (EBV) and its significance in nasopharyngeal carcinogenesis. Methods Forty nasopharyngeal carcinoma (NPC) biopsy tissues were used for detection of EBV BamHI f variant and LMP1 XhoI-loss by polymerase chain reaction (PCR), nested PCR, and RFLP (restriction fragment length polymorphism). Forty-eight samples of peripheral blood mononuclear cells (PBMC) taken from apparently healthy adult individuals were used for detection of LMP1 XhoI-loss. Three samples of amplified LMP1 exon 1 DNA from B95-8 cell line and 2 NPC tissues (one having XhoI-loss and the other having Wt-XhoI/XhoI-loss) were sequenced. Results Thirty out of the 40 NPC cases (30/40, 75%) harbored EBV BamHI f variant and the remaining 10 (10/40, 25%) harbored BamHI F prototype. Thirty out of the 39 NPCs (30/39, 76.9%) showed single EBV LMP1 XhoI-loss, 7 (7/39, 18.0%) showed single LMP1 Wt-XhoI (presence of a XhoI site in exon 1 of LMP1 gene, as in B95-8 cell line), and 2 (2/39, 5.1%) showed both LMP1 Wt-XhoI and XhoI-loss. Thirty-eight of the 39 NPCs (97.4%) showed EBV LMP1 XhoI-loss or/and BamHI F variant. In the NPC tissue (1 case only) showing the prototype of Wt-XhoI/BamHI “f”, there were several base substitutions, including 5 missense mutations and 2 silent mutations present in LMP1 exon 3, on DNA sequencing. On the other hand, 10 out of the 48 samples of PBMC taken from apparently healthy individuals could be amplified successfully by nested PCR for detection of LMP1 XhoI site. All of these 10 samples carried the prototype of EBV LMP1 Wt-XhoI. Conclusions The majority of EBV present in neoplastic cells of NPC is of BamHI “f” variant and/or possesses LMP1 XhoI-loss, as compared with that in healthy individuals. This genomic variation of EBV may bear some roles in the development and progression of NPC.