EB病毒诱发淋巴瘤的关键致瘤基因筛选、确定及功能研究

With hu-PBL/SCID chimeras constructed successfully and human-derived B-cell lymphomas induced by Epstein-barr virus(EBV), this project will introduce integration and information research strategy of systemic biology and make comparison of host genetic expression profiling and viral genetic expression profiling between human normal lymphocytes and EBV-induced lymphoma cells in SCID mice by detection using human whole genome expression profiling microarray and EBV genome expression profiling microarray. We will verify expression levels of those differentially expressed genes in more cases of lymphomas induced by EBV, which are before and after tumorigenesis of normal lymphocytes induced by EB virus using Real-time PCR technology, Western blot and immunohistochemistry staining. Through building the genetic expression mode and functional relationships of EBV-induced lymphomas, we will analyze and search the correlations between EBV gene expression profiling and host gene expression profiling, and find out some genes which play key roles in the process of tumorigenesis of human lymphocytes induced by EB virus. Then, we will further study the biological function of some key genes and their roles in the lymphomagenesis with experimental methods, such as gene transfection, RNA interference and so on, and with clinical specimen verification.

在已成功构建hu-PBL/SCID嵌合体小鼠并证实EB病毒诱发人源B细胞淋巴瘤的基础上，本课题引入系统生物学整体性、信息化的研究策略，分别采用人类全基因组表达谱芯片、EBV基因芯片检测比较<正常人淋巴细胞与SCID小鼠体内EBV诱发淋巴瘤>的宿主细胞基因表达谱和病毒基因表达谱；用实时定量PCR，Western blot，免疫组化染色等方法，验证检测EBV诱导人淋巴细胞肿瘤发生前/后的差异表达基因在多例诱发瘤中的表达水平。构建EBV诱发淋巴瘤的基因表达模式和功能关系，分析寻找EB病毒基因表达谱和宿主细胞基因表达谱之间的联系，筛查出EBV诱导人淋巴细胞发生肿瘤过程中起关键作用的某些基因。然后采用基因转染、RNA干扰等实验方法和临床标本验证，进一步研究确定其生物学功能及在淋巴瘤发生中的作用和意义。

本课题采用人类全基因组表达谱芯片检测比较正常人淋巴细胞、EBV转化淋巴母细胞与严重联合免疫缺陷动物(SCID小鼠) 体内EBV诱发淋巴瘤的宿主细胞基因表达谱；用实时定量PCR，Western blot，免疫组化染色等方法，验证EBV诱导人淋巴细胞肿瘤发生前/后的差异表达基因的表达水平。结果表明正常淋巴细胞和转化淋巴母细胞的宿主细胞基因表达模式存在明显差异，E2F1、PLK1、PTPN11、BIRC5、FYN等是EBV诱导淋巴细胞转化的关键基因/分子，其中E2F1， PLK1在细胞周期、细胞凋亡、端粒末端转移酶活性等途径中具有中枢作用。.采用RNA干扰、基因转染等实验方法和临床标本验证，确定PLK1 ，E2F1等基因的生物学功能及在淋巴瘤发生中的作用和意义。另一方面，将健康献血员EBV感染的人淋巴细胞移植到SCID小鼠体内，通过动物实验证实在SCID小鼠体内EBV可诱发产生人源性单克隆B细胞淋巴瘤，利用人类全基因组表达谱芯片检测EBV诱发淋巴瘤的宿主细胞基因表达谱，通过生物信息学分析，筛选出EGF，IL1B，CSF2，PBK和TLR2等可能是EBV诱发淋巴瘤的关键基因/分子。采用实时定量PCR和Western bolt 方法检测EBV诱发淋巴瘤细胞中EB病毒基因（3种LMPs 和6种EBNAs）的表达，结果发现LMP-1、LMP-2A、LMP-2B、EBNA-1、EBNA-3A在诱发淋巴瘤细胞中均表达上调。EBNA-2，EBNA-3B，EBNA-3C和EBNA-LP在EBV潜伏感染的正常人淋巴细胞中均不表达，在其发生的淋巴瘤细胞中阳性表达。.EBV主要通过促进细胞周期和阻碍宿主免疫功能诱导淋巴细胞转化和肿瘤发生。本课题研究结果提示EBV诱发淋巴瘤的细胞分子机制是一个多基因参与，多通路涉及、病毒基因与宿主细胞基因相互作用的过程。