NMHC IIA介导EB病毒感染鼻咽上皮细胞分子机制的研究

Epstein-Barr virus (EBV) has been causally associated with nasopharyngeal carcinoma (NPC). However, how the virus accesses nasopharyngeal epithelial cells (NPECs) has not been fully demonstrated. An efficient in vitro cell-free EBV infection system was established using premalignant NPECs via three-dimensional (3D) culture technique. Base on this model, we identified that non-muscle myosin heavy chain IIA (NMHC-IIA) is a receptor that promotes EBV infection of nasopharyngeal epithelial cells through Pull-Down, spectrometric analysis, Co-IP, siRNA and inhibitor experiment. However, the precise mechanisms of NMHC-IIA mediating EBV infecting nasopharyngeal epithelial cells need to be clarified. We will further testify NMHC-IIA as a receptor for EBV infecting nasopharyngeal epithelial cells using over-expression, antibody neutralization, co-localization methods; moreover, study endocytosis pathways and signal pathways of NMHC IIA mediating EBV entry into the cells using virus binding, internalization, cell fusion assay, immunofluorescence confocal microscopy and protein chip methods; finally, explore the relationship between the expression levels of NMHC IIA and the characteristics, prognosis of NPC patients utilizing immunohistochemistry. Together, these studies will try to elucidate the molecular mechanism of NMHC IIA mediating EBV infecting nasopharyngeal epithelial cells, which will help to provide theoretical clues for the research and development of drugs to cure NPC.

EB病毒与鼻咽癌密切相关。据报道非肌肉肌浆球蛋白重链IIA（NMHC IIA）是人类单纯疱疹病毒进入上皮细胞的受体。我们运用三维培养技术成功建立了稳定高效的游离EB病毒感染永生化鼻咽上皮细胞模型株；运用Pull-Down、质谱分析、Co-IP、siRNA沉默等方法，发现NMHC IIA是EB病毒感染鼻咽上皮细胞的受体，但其介导EB病毒感染鼻咽上皮细胞的机制尚不清楚。我们拟在此模型株中，采用病毒结合、内化、细胞融合、共定位和蛋白磷酸化检测芯片等技术，研究该受体介导EB病毒进入细胞可能的内吞途径和信号通路；在鼻咽癌组织标本中，采用免疫组化和原位杂交等技术探讨NMHC IIA的表达与EB病毒感染的相关性，结合患者临床病理资料和随访结果，试图阐明NMHC IIA介导EB病毒感染鼻咽上皮细胞的分子机制，寻找预防和治疗鼻咽癌的分子靶标，为预防和治疗鼻咽癌提供理论依据。

EB病毒主要感染B淋巴细胞和上皮细胞，与伯基特淋巴瘤、传染性单核细胞增多症和鼻咽癌密切相关。EB病毒感染B淋巴细胞的分子机制已清楚，但感染上皮细胞的分子机制还有待阐明。我们以稳定高效的游离EB病毒感染永生化鼻咽上皮细胞模型株为基础，创新性地发现NMHC IIA可以与EB病毒糖蛋白gH/gL的相互作用介导EB病毒结合到细胞表面，进一步采用抗体中和、共定位等方法确认了NMHC IIA是介导EB病毒感染鼻咽上皮细胞的受体。通过免疫组织化学染色，NMHC IIA在异常增生上皮细胞的膜上和细胞质中均有高表达，NMHC IIA表达与EB病毒感染异常增生上皮密切相关。采用表达谱芯片技术检测和分析EB病毒感染永生化鼻咽上皮细胞前后的差异基因和信号通路，发现EB病毒感染与actin结合细胞骨架的调节有关，NMHC IIA 是与actin结合的细胞骨架蛋白，进一步说明EB病毒感染鼻咽上皮细胞与NMHC IIA有关。我们应用兔多克隆抗体制备技术，制备抗NMHC IIA 的特异性抗体，为鼻咽癌的分子靶向治疗和研究NMHC IIA 在鼻咽癌中扮演的角色奠定基础。进一步分析表达谱芯片和转录组测序数据及一系列实验验证，我们创新性地发现RSAD2在鼻咽上皮细胞中具有抗EB 病毒天然免疫的作用；首次发现RSAD2除了抗EB病毒复制功能外，还具有促进鼻咽癌侵袭转移的生物学功能，为阐明EB逃逸宿主天然免疫机制提供实验依据，为鼻咽癌的防治提供新的思路和靶点。