鼻咽癌高风险人群鼻咽部游离EB病毒的鉴定及相关生物学行为研究

Epstein-Barr virus (EBV) plays an important role in nasopharyngeal carcinoma (NPC) observed in endemic regions. However, questions regarding the initial EBV on nasopharynx and its biological characteristics remain to be clarified. Some research groups including ours revealed there was a certain EBV DNA load in the nasopharyngeal brushing samples from healthy population in a case-control study. A significant elevation of EBV DNA load and positive rate was observed in high-risk population with positivity of VCA-IgA titer. Further experiments by EBERs in situ hybridization and transmission electron microscopy (TEM) revealed there were cell-free EBVs in the nasopharyngeal brushing samples. We hypothesize there are live cell-free EBV accumulated on the surface of nasopharynx in high-risk population, and they are prone to infect the epithelial cells. In this research, the people with high risk of NPC will be recruited and nasopharyngeal brushing samples will be collected. the EBV in the brushing samples will be separated and co-cultured with three kinds of host cell, in order to explore their ability of infection. The tropism will be identified by testing the key protein gp42 using western blot. The EBV load will be continuously monitored in a year. Our study provides a potential target in preventing nasopharyngeal carcinoma by blocking the EBV infection on nasopharynx in the future.

EB病毒感染与鼻咽癌发生密切相关，但鼻咽癌发生之前鼻咽部的EBV及其生物学特性是怎样的，这一源头上的关键问题尚不明确。我们在内的多个课题组的病例对照研究显示对照人群鼻咽部鼻咽刷样本中检测到EBV DNA，进一步的实验结果表明VCA-IgA抗体滴度阳性的高风险人群鼻咽部EBV DNA检测的阳性率和载量均显著高于抗体阴性的健康人群，并通过透射电镜直接在高风险人群鼻咽刷样本中观察到完整的EBV颗粒。因此我们推测鼻咽癌高风险人群鼻咽部聚集了具有感染性和鼻咽上皮细胞趋向性的游离EBV。围绕该假设，我们以鼻咽癌高风险人群为研究对象，拟通过进一步分离纯化鼻咽部EBV后与宿主细胞共培养，对EBV感染核心蛋白gH,gL及gp42的定量分析及连续的EBV载量的动态监测等明确鼻咽部EBV的行为特征，为后续以游离EBV为出发点，阻断EBV感染鼻咽上皮细胞这一关键环节，推动鼻咽癌的一级预防提供潜在靶点。

EB病毒感染与鼻咽癌发生密切相关，但鼻咽癌发生之前鼻咽部的EBV及其生物学特性是怎.样的，这一源头上的关键问题尚不明确。我们开展了三方面的研究并取得了重要研究结果：（1）通过成熟的鼻咽刷取样技术体系，对鼻咽癌患者和对照者鼻咽部EBV的构成来源进行了鉴定，通过检测EBV DNA 载量和EBV潜伏期或裂解期转录的特征性mRNA，发现鼻咽癌患者鼻咽刷中EB病毒主要来源于感染EBV的肿瘤细胞，但对照者鼻咽刷中的EB病毒主要来源于细胞外游离的EB病毒粒子，这为从源头上开展鼻咽癌的预防提供了理论基础。（2）在明确组成来源的基础上，提出EB病毒DNA甲基化在鼻咽癌患者和对照者鼻咽刷样本中会有显著不同的假设并进行了实验验证，结果显示对EB病毒特定CpG位点甲基化的分型检测，对鼻咽癌的诊断具有较好的准确度，尤其是在鼻咽盲刷取样条件下，敏感度依然达到89.7%，特异度达到97.6%，鼻咽盲刷的取样方式结合特定甲基化位点的分型检测这一技术方案为在鼻咽癌高发现场开展广泛的鼻咽癌筛查提供了可能。（3）开展了相关拓展研究，对EB病毒在鼻咽癌高发家系中和肺淋巴上皮瘤样癌中的特征突变序列进行了鉴定，并在前期基础上对鼻咽癌中异常表达的mir-483-5p的致癌机制进行了研究，这些结果对于揭示鼻咽癌的疾病机制具有积极意义，为针对性的开展鼻咽癌预防提供了更多潜在的靶点。