摘要
鼻咽癌是我国南方常见的恶性肿瘤，据WHO估计，80％的鼻咽癌发生在中国。鼻咽癌的病因学研究表明，鼻咽癌的发生是一个多因素，多阶段的过程，虽然遗传因素和环境因素与鼻咽癌的关系十分密切，但普遍认为EB病毒的感染是引起鼻咽上皮癌变过程的主要因素之一。然而EB病毒与鼻咽癌的关系还远未阐明，过去认为EB病毒在鼻咽癌中处于Ⅱ型潜伏感染状态，但随着分子生物学技术的不断改进，认为静止期鼻咽癌细胞也可能存在一定程度的裂解复制，但目前还没有直接的证据予以证实；其次EB病毒对鼻咽癌是致癌关系还是伴随关系也存在很大的争论，有必要进一步研究；再次哪些EB病毒瘤基因参与鼻咽癌的发生发展同样不清楚，潜伏膜蛋白LMP1是第一个被发现的ED病毒瘤基因，但尽管100％鼻咽癌细胞存在EB病毒基因组，却只有50-60％NPC组织表达LMP1，而缺乏LMP1表达的EB病毒也可以导致上皮细胞的增生转化，提示可能还存在另外的EB病毒瘤基因参与鼻咽癌的发生发展过程。BARF1基因位于EB病毒基因组BamHI-W区域，研究表明BARF1是EBV裂解期中的早期基因，该基因可促进入或猴肾上皮细胞永生化和恶性转化，而在EBV诱发B细胞转化中作用不大；在鼻咽癌中有高比例表达，而在淋巴系统肿瘤(除伯基特氏淋巴瘤外)中不表达，那么有可能是BARF1而不是LMP1在鼻咽上皮细胞癌变过程中扮演
中南大学 博士学位论文
更重要、更具普遍意义角色，提示有可能通过对 BARF的研究进一步
揭示EBV在鼻咽癌中的作用机制。
本研究应用荧光定量PCR技术检测同一个体的鼻咽癌、癌旁、鼻
咽癌病人相对正常鼻咽粘膜、血浆中EB病毒拷贝数及其他头颈部恶性
肿瘤、正常人鼻咽粘膜、原代培养鼻咽癌细胞、B95十细胞系及CNEI
细胞系中EB病毒拷贝数；并采用原位杂交技术对同一个体的鼻咽癌、
癌旁、鼻咽癌病人相对正常鼻咽粘膜组织中的EB病毒进行定位检测；
同时应用巢式干CR技术检测鼻咽癌、癌旁。相对正常鼻咽粘膜、正常
人鼻咽粘膜及头颈部其他恶性中BARFI基因的表达情况；将鼻咽癌、
癌旁、鼻咽癌病人相对正常鼻咽粘膜 BARF基因的表达强度与对应样
本的EB病毒拷贝数进行等级相关性分析。现将研究结果报告如下：
1、慢性鼻咽炎的鼻咽粘膜用病毒检出率为 80＊门八 人 EBV平
均拷贝数为 6．7 XIOy呷DNA；鼻咽癌病人的相对正常鼻咽粘膜 EB病
毒检出率为72．弧（3外47）BBV平均拷贝数为9．8 X my卜咄 NANA；二者
之间感染EB病毒的机率、潜伏感染EB病毒的数量没有显著性差异。
提示无论是鼻咽癌病人还是健康人群EB病毒在鼻咽组织中的潜伏感染
是一种普遍现象。应用EBERl探针原位分子杂交发现鼻咽癌病人相对
正常粘膜上皮细胞未检测到EBERl的表达信号，而在癌旁靠近癌一侧
的不典型性增生细胞及所有癌组织中的癌细胞均有EBERl 的表达信
号，提示EB病毒最初并不感染正常的上皮细胞，而是由于某种因素才
导致用病毒进人鼻咽上皮细胞。
2、鼻咽癌EBV平均拷贝数为6．9 XIO丫V＊NA：癌旁BBV平均拷
二二
中南大学 博士学位论文
贝数为 1．SX 10’／卜gDNA；才对正常粘膜为 9．SX 10‘／pgDNA；鼻咽癌
组织及其癌旁、相对正常粘膜三者之间；鼻咽癌与癌旁之间；癌旁和
相对正常粘膜之间EB病毒拷贝数存在显著性差异。其他头颈部恶性肿
瘤mV出率为 61．9％（13／21），平均拷贝数为 1．2 X 10‘／卜卯NANA。说明
鼻咽癌细胞中EB病毒存在有限的裂解和复制，而鼻咽癌病人鼻咽相对
正常粘膜、其他头颈部恶性肿瘤潜伏EB病毒不进行裂解复制增殖，提
示EB病毒从一种潜伏感染状态（不进行裂解复制增殖）转变到另一种
潜伏感染状态（有限的裂解和复制状态）可能是EB病毒致鼻咽癌的重
要环节。
3、鼻咽癌病人血浆中游离 EB病毒检出率为 93．6％H4竹7人 临
床 Ill期病人血浆游离 EBV平均拷贝数为 1．IXIO丁呷NA，Ill IV期
病人血浆游离 EBV平均拷贝数为 7．8 XIO丫收DNA，二者之间血浆游离
EB病毒拷贝数存在显著差异；原代培养鼻咽癌细胞第4、5、6代细胞
EB病毒拷贝数逐渐减少，第 10代不能检测到 EB病毒；说明鼻咽癌细
胞中EB病毒存在不断丢失现象，提示EB病毒在鼻咽癌细胞各期都可
能存在一定程度的复制以维持EB病毒在鼻咽癌细胞中持续感染。
4、BARF基因在鼻咽癌、癌旁、相对正常粘膜中的表达率分别为
91．5％（43／47）、46．8％（22／47）、6．4％（3／47）；其他头颈部恶性月瘤
均未检测到BARFI基因表达；提示启动BARn基因的表达有可能是用
病毒致鼻咽癌的关键事件之一。
5、鼻咽癌、癌旁、鼻咽癌病人相对正常鼻咽粘膜的EB病毒拷贝
数与BARFI基因的表达强度存在明显的等级相关性；携带EB病毒并存
互五二
中南大学 博士学位论文
在一定程度裂解复制的B95上细?
Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China. WHO estimates that NPC cases are much higher in China, being 80% of cases of the world. The etiology study of NPC indicated that the development of the NPC is a process with multi-factors and multi-stages. Heredity and environment are related to development of NPC, however, It was reported that infection of Epstein-Barr virus (EBV) is one of the major factors that activate malignant transformation of the nasopharyngeal epithelial cells. But the mechanism of reaction between EBV and NPC is not clear yet. It was believed that EBV of NPC exists in infectious status II, but with the development of molecular biology, recently studies showed that lytic cycle was likely appeared in EBV patients with static NPC cells. However, this has not been elucidated yet. Besides, there has been dispute over whether EBV induces NPC or accompanies NPC. Finally, it is unknown which gene of EBV is involved in the development of NPC. Although EBV appears in all cells of NPC, one of the EBV genes, latent membrane protein (LMP1) only showed 50%-60% of expression in NPC patients, and EB without LMP1 expression might also induce malignant transformation of the
nasopharyngeal epithelial cells, suggesting that other EBV genes might be involved in development of NPC. BamHl-A right frame 1 (BARF1), Located in BamHl-W region of EBV gene, is an early stage gene in lytic cycle. Studies reported that It might have ability to immortalize human or monkey kidney epithelial cells and induce tumors. This gene, however, has been tested that has little effect on inducing B lymphocyte transformation. BARF1 other than LMP1 may be important in inducing malignant transformation in nasopharyngeal epithelial cells since BARF1 showed highly expression in NPC cases, and no expression in lymphoma except for Burkitt's lymphoma. These findings suggest that further study on BARF1 may ascertain the mechanism of EBV in development of NPC.
In this study, Carcinoma tissues, adjacent tissues, and normal Nasopharyngeal tissues from 47 patients with nasopharyngeal poorly differentiated squamous cell carcinoma (NPDSCC) prepared simultaneously were tested for the presence of the EBV DNA copies levels using real-time quantitative Polymerase Chain Reaction (PCR) assays. As control, carcinoma tissues were obtained form 21 head and neck carcinoma (HNC) patients, and nasopharyngeal tissues from another 10 healthy subjects for detection of EBV DNA copies. Original culture NPC cells, cell line B95-8 and cell line CNEl were also used as control groups to test their EBV DNA copies levels. Besides, we used in situ
hybridization for in situ detection of EBV in carcinoma tissues, adjacent tissues, and normal nasopharyngeal tissues of patients with NPDSCC. Furthermore, BARF1 expression levels of carcinoma tissues, adjacent tissues, and normal Nasopharyngeal tissues of NPC patients, as well as healthy nasopharyngeal tissues and other HNC tissues were detected by using nested PCR. Lastly, the relationship between EBV DNA copies and BARF1 of carcinoma tissues, adjacent tissues, and normal nasopharyngeal tissues of NPC patients was identified using rank correlation analysis.
The results of this study showed that:
1. 80% of nasopharyngeal tissues in chronic nasopharyngitis patients and 72.3% of normal nasopharyngeal tissues of NPC patients (34/47) were positive for EBV DNA with mean copies of 6.7+102/gDNA and 9.8+103/gDNA, respectively. There was no significant difference on infectious rate and infectious level of EBV between these two groups. These results indicate that EBV infection appeared in both NPC and healthy subjects. Using EBER-I in situ hybridization, EBER-I expressive signal was not detected in normal nasopharyngeal epithelial cells of NPC, but found in non-proliferation cells in adjacent tissues and in carcinoma cells of NPC, suggesting that a factor might contribute to the entrance of EBV into nasopharyngeal epithelial cell.
2. The means of EBV DNA in carcinoma tissues, adjacent tissu
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