EBV阳性鼻咽癌细胞的外泌体对鼻咽癌淋巴结转移的影响与机制研究

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in southern provinces of China and often accompanied by significant lymph node metastasis, its morbidity and mortality rate is the top rank of malignant tumors. EBV infection is often associated with nasopharyngeal carcinoma cells, but the role and mechanism of EBV in lymph node metastasis of nasopharyngeal carcinoma remain unclear. Role of exosomes in the metastasis of tumors has been gained more and more attention. Our previous studies have found that EBV-positive NPC exosomes promote lymphatic endothelial vascularization and migration in vitro and local metastasis in vivo, it is suggested that EBV positive nasopharyngeal carcinoma derived exosomes may be closely related to lymph node metastasis of nasopharyngeal carcinoma. In this project, the regulation of EBV-positive nasopharyngeal carcinoma exosomes on lymphocyte endothelial cells.vascularization and migration, lymphatic angiogenesis and lymph node metastasis was first identified with in vivo and in vitro experiments, and then the effects of nucleic acids and proteins in exosomes were compared to determine the important molecular types to be studied, the proteins or nucleic acids were analyzed by mass spectrometry or sequencing. To further clarify the molecular mechanism of EBV-positive nasopharyngeal carcinoma exosomes. The study provides an effective and promising theoretical basis for clinical application of combine anti-lymphangiogenesis drugs and radiotherapy for nasopharyngeal carcinoma. it is beneficial for improving the survival rate of nasopharyngeal carcinoma patients.

鼻咽癌是我国南方省份常见的恶性肿瘤之一且具有显著的淋巴结转移，其发病率与致死率均位于恶性肿瘤前列。鼻咽癌细胞常伴随EBV感染，但EBV在鼻咽癌淋巴结转移中的作用与机制还不明确。外泌体在肿瘤转移中的作用逐渐受到人们的重视。前期研究发现：离体条件下， EBV阳性鼻咽癌细胞外泌体促进淋巴内皮细胞成管与迁移；在体条件下，其可促进鼻咽癌局部转移。提示EBV阳性鼻咽癌细胞外泌体可能与鼻咽癌淋巴结转移密切相关。本项目首先通过体内与体外实验明确EBV阳性鼻咽癌细胞外泌体对淋巴内皮细胞成管与迁移、淋巴管新生以及鼻咽癌淋巴结转移的调控作用，进而比较EBV阳性鼻咽癌细胞外泌体内核酸与蛋白的作用大小，确定发挥重要作用的分子类型，利用质谱或测序分析蛋白或核酸，进一步明确发挥作用的分子和分子机制。本研究为临床联合应用抗淋巴管新生药物和放疗治疗鼻咽癌提供有效且具应用前景的理论依据，对提高鼻咽癌患者生存率具有重要意义。

研究背景 EB病毒（EBV）促进鼻咽癌淋巴结转移，淋巴管新生是肿瘤淋巴结转移的关键步骤，但EBV阳性鼻咽癌细胞外泌体对淋巴管新生与鼻咽癌淋巴结转移的影响还不清楚。.研究内容 利用超速离心获得外泌体，对外泌体进行免疫印迹与纳米颗粒追踪分析（NTA）鉴定。利用PKH67染料吞噬实验，验证外泌体可以被淋巴管内皮细胞摄取。离体条件下，分别利用等体积培养基、永生化鼻咽上皮细胞（NP69）、NPC43、C666-1细胞外泌体和除去外泌体的NPC43与C666-1上清蛋白处理淋巴管内皮细胞，检测淋巴管内皮细胞成管与迁移情况；在体实验，首先建立裸鼠腘窝淋巴结转移模型，负瘤裸鼠被随机分成4组，3只/组。对照组：腘窝处注射生理盐水；NP69外泌体组：腘窝处注射NP69外泌体；NPC43除去外泌体上清组；腘窝处注射NPC43除去外泌体上清蛋白；NPC43外泌体组：腘窝处注射NPC43外泌体。每2天注射1次，共4次。检测腘窝淋巴结肿瘤转移情况与腘窝淋巴结大小。提取NPC43外泌体内核酸，转染淋巴管内皮细胞，检测淋巴管内皮细胞的迁移与成管变化，对外泌体内核酸进行测序分析，寻找差异的miRNA进行体内外功能验证。.研究结果 超速离心获得外泌体富含外泌体相关蛋白，同时NTA鉴定表明其粒径范围正常。鼻咽癌细胞与NP69外泌体可以被淋巴管内皮细胞摄取。NPC43与C666-1外泌体较空白对照组与NP69外泌体可显著促进淋巴管内皮细胞成管与迁移（P<0.05），NPC43与C666-1外泌体组与对应除去外泌体上清蛋白组间无显著性差异（P>0.05）；腘窝淋巴结转移实验发现：NPC43外泌体较空白对照组、NP69外泌体以及除去外泌体的NPC43上清蛋白可显著促进鼻咽癌细胞淋巴结转移以及局部淋巴管新生（P<0.05）。分析出外泌体内具有差异的miRNA分子，备选分子的功能实验还在验证中。.关键数据 明确EBV阳性鼻咽癌细胞外泌体可以通过刺激淋巴管内皮增殖进而促进淋巴管新生与鼻咽癌淋巴结接转移。.科学意义 外泌体内效应分子可为鼻咽癌淋巴结转移的治疗提供潜在治疗靶点，血清中外泌体组分中的分子物质可作为鼻咽癌淋巴结转移的潜在分子标物。