CLEC4M 通过细胞接触模式介导EB病毒感染鼻咽上皮细胞的机制研究

Epstein-Barr virus (EBV), also known as type 4 human herps virus (HHV4), is a ubiquitous virus and a pathogenic factor related to various epithelial and lymphocytic malignant tumors. About 90% adults have been infected by EBV. However, the mechanism in which EBV infects and transforms human epithelial cells is still not clear. Our preliminary studies showed that the efficiency of EBV infection of epithelial cells significantly increased when they were co-cultured with EBV-positive lymphoma cells, in which expression of a molecule, namely CLEC4M (C-type lectin domain family 4 member M) were up-regulated. Interestingly, our study also showed that EBV infection efficiency of epithelial cells decreased significantly after addition of mannan, which acted as a competitive antagonist for CLEC4M. CLEC4M has been shown to correlate closely with infection of various pathogenic microorganisms, such as HIV, HCV. Bases on previous published reports and our preliminary studies, we hypothesize that CLEC4M is also linked to EBV infection of epithelial cells. Therefore, in this study, we plan to investigate the exact role of CLEC4M, as an affinity receptor on cell memberane, in the EBV infection of nasopharyngeal epithelial cells using the methods including cell co-culture, gene editing, Co-Immunoprecipitation, immunoelectron microscopy,etc. We also plan to investigate the different roles of Raji and KMH2 cell models carrying different tandem repeat variations of CLEC4M neck region in EBV infection efficiency, and study the possible pathways involving functional and structural interactions between CLEC4M and EBV glucoproteins and epithelial cell membrane proteins. This study would provide a deeper insight into the rules of occurrence and development of EBV-related diseases, laying a good foundation for their early warning and diagnosis.

EB病毒（EBV）是一种普遍存在的人类4型疱疹病毒（HHV4），是多种上皮及淋巴源性恶性肿瘤的病原因子，其感染上皮细胞并致其恶性转化的机制仍未阐明。前期研究发现与淋巴瘤细胞系（EBV+）共培养后，上皮细胞感染EBV的效率明显升高，而淋巴瘤细胞CLEC4M基因表达水平明显上调，加入其竞争性拮抗剂后上皮细胞感染效率明显下降。CLEC4M 是C型凝集素家族4，膜蛋白M，多个研究证实其与多种病原微生物感染有关。结合本课题组的前期研究结果，我们推测其亦参与了EBV感染上皮细胞的机制。因此，本课题拟通过细胞共培养模型、基因编辑等实验验证CLEC4M作为细胞膜表面亲和性受体促进EBV感染鼻咽上皮细胞的功能，同时分析其不同颈区重复序列变异在介导EBV感染中的差异，再通过蛋白互作研究其与EBV糖蛋白及上皮细胞膜蛋白的关系，进而分析其作用途径，为进一步阐明EBV相关疾病的发生发展规律及早期预警奠定基础。

EB病毒感染是鼻咽癌发生发展的重要机制，其感染上皮细胞并致其恶性转化的机制仍未阐明。研究发现C型凝集素家族4，膜蛋白M，(CLEC4M)，具有参与抗原提呈、介导多种病原微生物感染的作用，其颈区重复序列片段长度与蛋白质的寡聚化及抗原识别密切相关。本课题组前期研究发现广西人群中CLEC4M颈区重复序列为9次重复的个体患鼻咽癌的风险比其他多态性亚型要高。目的：本研究构建7和9颈区重复序列的CLEC4M慢病毒表达载体，分别感染鼻咽癌细胞HONE1、鼻咽部永生化细胞系NP69、淋巴瘤细胞系Raji，以及淋巴管内皮细胞系HLEC，通过直接EBV感染及细胞共培养感染模型验证CLEC4M介导EBV感染的作用及不同颈区重复片段差异对EBV易感性产生的影响，并研究CLEC4M介导的细胞间粘附作用对肿瘤侵袭、迁移的影响。结果：EBV及空载慢病毒感染健康志愿者外周血单个核细胞PBMC及HLEC细胞后CLEC4M表达上调，过表达CLEC4M可以提高HLEC的EBV直接感染效率，特别是颈区为9重复片段的CLEC4M，但对HONE1及NP69的直接EBV感染效率无明显促进作用。在HLEC和Raji细胞中上调CLEC4M表达，可通过细胞介导模式促进EBV感染鼻咽癌细胞HONE1，但对NP69无明显影响。CLEC4M上调的HLEC细胞对鼻咽癌5-8F、6-10B细胞的粘附、趋化作用增强。纯化的CLEC4M蛋白亦可以粘附于5-8F和6-10B细胞，可用流式细胞仪检出，特别是具有高淋巴结转移潜能的5-8F细胞与CLEC4M粘附作用更明显。结论：CLEC4M主要通过细胞间粘附趋化作用，利用HLEC细胞或淋巴细胞介导的细胞-细胞接触模式，促进EBV感染鼻咽癌上皮细胞，其中颈区为9重复片段的CLEC4M作用较强；正常鼻咽部上皮细胞对EBV不易感,恶性转化可能是EBV感染上皮细胞的前期事件。