定量和定位检测EB病毒在鼻咽癌组织中的感染状态

Quantitative and Localized Detection of Epstein-Barr Virus Infectious Status in Nasopharyngeal Carcinoma Tissue

背景与目的EB病毒(Epstein-Barrvirus,EBV)与鼻咽癌密切相关,但EBV是鼻咽癌的致瘤因素还是仅仅是伴随关系有待进一步研究,而且EBV在鼻咽癌发生过程中的潜伏状态也不明确,本研究通过检测EBV在不同鼻咽组织中的表达变化,为进一步阐明EBV与鼻咽癌发生的关系提供线索。方法应用荧光定量PCR(real-timePCR)技术定量检测47例鼻咽低分化鳞癌患者癌、癌旁及相对正常鼻咽粘膜组织中的平均EBV拷贝数;并采用原位杂交技术(地高辛标记的EBER-1探针)对鼻咽癌、癌旁、对侧正常鼻咽粘膜中的EBV进行定位检测。以10例正常人鼻咽粘膜组织作对照。结果正常人鼻咽粘膜EBV检出率为80%(8/10),平均拷贝数为6.7×102/滋gDNA;100%(47/47)鼻咽癌和癌旁组织以及72.3%(34/47)的对侧正常鼻咽粘膜组织检出EBV,平均拷贝数分别为6.9×105/滋gDNA、1.5×105/滋gDNA、9.8×103/滋gDNA。正常人鼻咽粘膜、鼻咽癌患者对侧正常鼻咽粘膜之间感染EBV的几率、潜伏感染EBV的数量没有显著性差异(P>0.05);鼻咽癌及其癌旁、对侧正常粘膜组织三者之间,鼻咽癌与癌旁组织之间,癌旁和对侧正常粘膜组织之间EBV拷贝数存在显著性差异(P<0.01)。EBER-1原位分子杂交发现,对侧正常粘膜上皮细胞未检测到EBER-1的表达信号,癌旁靠近癌一侧的部分不典型增生细胞检测到EBER-1弱阳性信号,而在癌组织中的所有癌细胞均有EBER-1强阳性信号。结论无论是鼻咽癌患者还是健康人,EBV在鼻咽组织中的潜伏感染是一种普遍现象,但从鼻咽癌患者对侧正常鼻咽粘膜到癌旁再到癌组织EBV感染的量发生了改变,说明鼻咽上皮细胞中EBV感染量和表达信号的改变很可能是EBV致鼻咽癌的重要环节。

BACKGROUND & OBJECTIVE: Epstein-Barr virus (EBV) closely relates to nasopharyngeal carcinoma (NPC). Whether EBV is a causative viral agent or a by-stander in the carcinogenesis of NPC needs further study. The latent status of EBV during carcinogenesis of NPC remains unknown nowadays. This study was to explore the infectious status of EBV in NPC, and analyze the correlation of EBV to carcinogenesis of NPC. METHODS: Carcinoma tissues,adjacent tissues, and contralateral normal nasopharyngeal tissues from 47 patients with poorly-differentiated nasopharyngeal squamous cell carcinoma were prepared simultaneously. Nasopharyngeal tissues from 10 healthy subjects were used as control. EBV DNA copies levels in the specimens were tested using real-time quantitative polymerase chain reaction (PCR); localization of EBV was detected by in situ hybridization [digoxigenin-labeled EBV-encoded RNA 1 (EBER-1) probe]. RUSULTS: Infectious rate of EBV was 80.0% in nasopharyngeal tissues of healthy subjects (mean EBV DNA copies of 6.7×102/?滋g DNA), 100% in carcinoma tissues and adjacent tissues of NPC patients, and 72.3% in contralateral normal nasopharyngeal tissues of NPC patients (mean EBV DNA copies of 6.9×105/?滋g DNA, 1.5×105/?滋g DNA, and 9.8×103/?滋g DNA, respectively). There was no significant difference in infectious rate and level of EBV between controlateral normal nasopharyngeal tissues of NPC patients and nasopharyngeal tissues of healthy subjects (P>0.05); infectious level of EBV was significantly higher in carcinoma tissues than in adjacent tissues (P<0.05), and significantly higher in adjacent tissues than in contralateral normal nasopharyngeal tissues of NPC patients (P<0.01). Using in situ hybridization, EBER-1 expression signal was not detected in contralateral normal nasopharyngeal epithelia of NPC patients, weak in atypical hyperplastic cells in adjacent tissues, and strong in carcinoma cells. CONCLUSIONS: EBV latent infection is a common phenomenon in nasopharyngeal tissues of both NPC patients and healthy persons. The enhancement of EBV infection plays an important role during carcinogenesis of NPC.