鼻咽脱落细胞EB病毒载量及其潜伏/裂解基因表达分布规律与鼻咽癌发生的关联研究

Previous studies and our preliminary research indicated that the Epstein-Barr virus (EBV) in nasopharyngeal tissue may constitute a key event in nasopharyngeal carcinoma (NPC) carcinogenesis. The disparity of viral DNA loads in nasopharynx between populations from southern and northern China, could be related to the extremely disparity of NPC incidence in these areas. Moreover, certain environmental factors may reactivate EBV in body and then increase the risk of NPC. Therefore, the critical points for NPC prevention were to further investigate the potential modifiable risk factors of NPC, including EBV infection status in nasopharynx and the environmental factors influencing EBV infection, and to identify specific molecular signatures for recognizing high-risk individuals. Based on our established convenient, and painless method of nasal brush sampling, we plan to conduct large-sample-sized molecular epidemiological studies detecting DNA loads and crucial viral latent/lytic gene expression in nasal exfoliated cells. Analyses will be performed to explore the distribution of EBV DNA loads and specific gene expression in NPC case-control populations and healthy populations in NPC endemic area and non-endemic area. The difference of viral load and gene expression between these populations was also analyzed. In addition, individual exposure for environmental factors (smoking, alcohol, Cantonese food, et al.) and cytokines like IL-12 in nasal cavity which may affect the infection status of EBV will be investigated. Crucial indicators such as EBV DNA loads and certain gene expression in nasal exfoliated cells and its environmental factors of NPC high-risk individuals will be identified. As far as we know, this would be the first study to break through the bottleneck of nasopharynx sampling method in epidemiological study, to focus on the direct biological changes in lesion location, and to detect viral biomarkers in nasal exfoliated cells. Our results will provide important epidemiological evidence for building effective way in prevention of NPC.

既往研究提示，鼻咽组织中的EB病毒载量增高可能是鼻咽癌关键病因学改变，其在我国南北方人群中分布的差异可能与鼻咽癌地区发病差异有关;且环境因素可通过激活EB病毒作用于鼻咽癌发生。目前，进一步发现EB病毒感染状况及环境因素等可改变的病因，寻找特异性分子指标更准确鉴别高危人群是鼻咽癌预防领域的研究难点。本项目将利用我们已建立的，快捷无创的鼻咽刷采样法采集鼻咽脱落细胞，开展鼻咽部EB病毒载量、病毒关键裂解/潜伏期基因表达的大样本量分子流行病学研究，探索其在鼻咽癌病例对照人群、高低发区健康人群中的分布规律和人群差异，并探索分析环境暴露、鼻咽腔微环境对鼻咽EB病毒感染情况的影响；发现高危人群鼻咽部EB病毒载量、潜伏/裂解感染规律及内外环境因素的影响。该研究首次突破流行病学研究的鼻咽取材瓶颈，锁定病灶部位的直接生物学改变，研究鼻咽脱落细胞EB病毒分子标志物，为建立鼻咽癌有效预防措施提供重要流行病学证据。

以鼻咽部EB病毒载量和特定血清EB病毒抗体升高为标志的EB病毒激活是鼻咽癌发生早期的关键病因学事件，我们探寻了可能影响EB病毒激活的内外环境因素：基于鼻咽癌高发区健康人群，使用SOLAR分析，对影响鼻咽部EB病毒载量的因素进行了整体分析，发现遗传是影响其水平的主要因素，遗传度可达40%以上；发现个体生活压力事件如长期严重受“家庭经济困难”影响可能和鼻咽部EB病毒载量升高有一定关联。我们针对EB病毒不同的潜伏/裂解期蛋白抗体（Zta-IgA、VCA-IgA、EBNA-1-IgA、LMP1-IgA）水平开展宿主遗传因素对其的影响，发现携带特定的HLA等位基因型DRB1*09:01等，和裂解期关键蛋白Zta的抗体水平具有显著的关联（显著性达Bonferroni校正水平，OR=1.80, 95% CI=1.32-2.45; P =1.82×10-4），联合吸烟分析发现，与携带非风险型等位基因的个体相比，轻度吸烟者并携带风险型等位基因者的Zta抗体阳性风险显著增高（OR=2.19，95%CI=1.15-4.15）；携带风险等位基因的重度吸烟者风险增加了2.7倍（95%CI=2.01-6.80），而非携带该等位基因的重度吸烟者OR值仅为1.72（95%CI=1.02-2.90），提示携带特定等位基因的个体如果联合其他危险因素，可能使其发生EB病毒激活的风险更高。此外，作为当前鼻咽癌早期最为敏感的标志物之一的VCA抗体，其显著受遗传因素影响，遗传度为33.33%；校正可能的影响因素后，受“家庭经济困难”重度影响者的VCA抗体阳性风险是不受影响者的2.36倍（95%CI=1.06-5.28）。我们还探索从EB病毒本身变异去寻找影响其激活的因素，通过全球EB病毒关键蛋白序列进化分析和种群结构分析，鉴定并验证了4个和华南鼻咽癌特异相关的EB病毒变异，累积4个变异患鼻咽癌的风险是无变异者的32倍（95%CI=9.18-111.49），其中BNRF1 V1222I为首次报道，再经独立人群验证依然和鼻咽癌显著相关（OR=5.55，95%CI=3.22-9.55），但其和EB病毒激活的关联还需进一步证实。本项目从多角度发现了影响鼻咽癌相关的EB病毒激活事件的内外因素，对于研究鼻咽癌的病因、鉴定高危人群开展预防措施有一定意义，已发表SCI论文5篇，达到项目研究目标。