鼻咽癌血浆外泌体的EB病毒DNA特征及来源研究

EBV has been associated with the development of nasopharyngeal carcinoma (NPC), and plasma EBV DNA is currently considered to be the most attractive potential biomarker to guide individualized treatment of NPC patients. However, little information is available regarding the molecular characteristics and origin of plasma EBV DNA. Some researchers considered that therapy-induced cancer cell death or apoptosis is the origin of plasma EBV DNA, due to observing an initial rise in plasma EBV DNA levels after treatment. Nevertheless, our research team found that the mechanism of origin of plasma EBV DNA mentioned above cannot elucidate some phenomenon that we observed in clinical practice, such as no initial rise in plasma EBV DNA levels after treatment, fluctuations of plasma EBV DNA during the treatment and the undetectable plasma EBV DNA for some of local and regionally advanced NPC before, during and after treatment, even with EBV-encoded RNAs by in situ hybridization positive (Eber+). Interestingly, the plasma EBV DNA was detectable in the exosomes derived from the plasma of NPC patients by Real-Time Quantitative PCR, according to our pre-experiment data. Therefore, we made a hypothesis that the plasma EBV DNA may partially origin from exosomes. Thus, this study is going to detect the plasma EBV DNA levels of exosomes and supernatant from different cells, and explore the status of EBV DNA between exosomes and supernatant by the method of EBV Capture Sequence, elucidated the mechanism of origin of plasma EBV DNA and made the plasma EBV DNA better use in clinical practice.

鼻咽癌发生发展与EB 病毒相关。血浆EBV DNA是指导鼻咽癌个体化治疗的潜在分子标志物，但其分子特征及来源仍不清楚。部分学者认为血浆EBV DNA最有可能来自坏死和凋亡的癌细胞。但是，课题组前期发现该来源机制不能解释血浆EBV DNA的多种临床现象，如肿瘤在放化疗后迅速缩小，血浆EBV DNA并没有迅速上升；治疗过程中血浆EBV DNA上下反复波动等；同时我们前期的预实验在患者血浆中的外泌体中检测出了EBV DNA，由此我们推断血浆EBV DNA可能部分来源于外泌体。因此，本研究将通过Real-time PCR检测不同细胞分泌的外泌体和分离外泌体后上清液EBV DNA的拷贝数，再采用EBV 基因的捕获测序的方法探究外泌体和上清液中DNA的存在形式，明确血浆EBV DNA来源的细胞类型（癌细胞或其它细胞）以及细胞状态（活细胞或死亡细胞），将为血浆EBV DNA更好的运用于临床提供理论基础。

鼻咽癌是一种高发于我国华南地区的上皮源性恶性肿瘤，EB病毒感染是认为与鼻咽癌发生发展最为密切的相关因素。虽然目前血浆EBV DNA在鼻咽癌的临床运用中具有重要的价值，但其分子特征及来源的具体机制仍不清楚。本研究拟在肿瘤细胞本身、淋巴细胞及巨噬细胞通过主动释放外泌体方向进一步探讨血浆EBV DNA 来源的生物学机制；另一方面从临床出发，进一步确证血浆EBV DNA在指导鼻咽癌个体化治疗方面的重要作用。本课题组成功分离及提取血浆及细胞培养上清液中的外泌体及外泌体中的DNA，并进行了外泌体的鉴定；通过大样本临床数据分析证实EBV DNA可作为指导鼻咽癌个体化同期放化疗的重要工具。为血浆EBV DNA指导鼻咽癌的个体化同时放化疗提供前期的理论基础，使血浆EBV DNA能够更广泛地运用于临床。