弥漫大B细胞淋巴瘤中LMP1癌基因下调B细胞终末分化调控因子Blimp1基因的作用机制及预后预测研究

Diffuse large B cell lymphoma is a group of a significant clinically heterogeneous tumor.EBV positive DLBCL tumor involved younger patient has highly aggressive behavior and highly mortality, but not to be classify, now. EB viral complete replicate cycle in B lymphocyte into plasma cell differentiation,Blimp1 transcript is an important regulator factor in B lymphocyte into plasma cell differentiation. Study has discovered DLBCL with high-frequency Blimp1 gene mutation which associated with poor prognosis in China. EBV participate tumorigenesis throughout to dowm regulator Blimp1 transcript is still unclear, Different molecular subtype DLBCL existing Blimp1 gene differential expression and associated signaling pathway molecular differential expression whether or not has not been systematically studied in Xinjiang.Combining detection Blimp1 and LMP1 gene mutation and differential expression possibly effective predict the tumor's prognosis;we will accurately category molecular subtypes using gene chip technology and screen Blimp1 and LMP1 gene differential expression between different subtypes DLBCL; We will validate the differential expression gene such as Blimp1 and LMP1 gene in mRNA level using Chromatin immunoprecipitation coprecipitation - real-time quantitative PCR technology, than we will detect the differential gene above metioned using fluorescence quantitative qRT-PCR method in mRNA expression levels, than we will detect Blimp1 and LMP1 gene mutation with PCR combined direct sequencing method, MSPCR method will detect two gene promoter methylation levels, and anlysis Blimp1 and LMP1 biology function with siRNA method, than we will combine clinicopathological factors to establish a multi-gene prognostic predict model, and to lay the foundation for the implementation of individualized treatment, improve and monitor the therapy efficacy, reveal oncogenic pathways that are used differentially by the DLBCL subtypes, reinforcing the view that they represent pathogenetically distinct diseases.

DLBCL是一组具显著临床异质性肿瘤，EBV+DLBCL累及年轻患者则具更强侵袭性和更高致死率。EBV在B细胞终末分化阶段进行复制发挥致瘤作用，Blimp1是该阶段重要转录因子。研究发现中国人DLBCL存在高频Blimp1基因突变，且与预后密切相关。癌基因LMP1是EBV相关肿瘤潜伏感染的客观指标，能以多种机制促进肿瘤形成和发展，LMP1在DLBCL中是否通过下调Blimp1基因而增强其致瘤性？两基因间的相互作用机制及表达差异和突变模式能否有效预测肿瘤预后？拟用基因芯片技术筛选不同分型间差异表达的Blimp1、LMP1基因；染色质免疫共沉淀-定量RT-PCR验证芯片检测结果，PCR联合直接测序法检测上述基因突变模式； MSPCR方法检测两基因启动子区甲基化水平，研究两基因作用机制。应用siRNA干扰技术分析两基因生物学功能，为患者提供个体化治疗方案，提高和监控疗效，阐述肿瘤发生机制奠定基础

弥漫性大B细胞淋巴瘤（Diffuse large B cell lymphoma，DLBCL）是成人最常见的非霍奇金淋巴瘤（NHL），国内淋巴瘤协作组统计 DLBCL 发病率高达 40%-50%，高于国际 25%-30%的发病率。目前临床上应用国际预后指数（IPI）评分评估DLBCL患者预后，但即使是IPI评分相同的患者，其预后也不尽相同，寻找DLBCL预后判断的相对特异的分子指标迫在眉睫。研究发现中国人DLBCL存在高频PRDM1基因突变，且与预后密切相关。研究发现Blimp-1可抑制C-MYC基因表达。Blimp1的表达可诱导B细胞分化成浆细胞并通过结合C-MYC启动子中的位点抑制C-MYC，使细胞停止分裂。基于以上研究，我们推测PRDM1/Blimp1的缺失可导致B细胞过度增殖，从而参与B细胞淋巴瘤的形成。本研究拟用免疫组织化学（IHC）、逆转录聚合酶链反应（RT-PCR）、甲基化特异性PCR（MSP）及体外小干扰RNA（siRNA）转染DLBCL细胞系沉默PRDM1基因后观察C-MYC表达情况，从蛋白水平观察DLBCL中Blimp1、C-MYC的表达情况及甲基化状态，探讨PRDM1基因失活对C-MYC的调控，同时着重观察两基因与免疫分型、临床病理参数及预后相关性，探讨该基因失活在DLBCL发生发展中的作用。为今后DLBCL发病相关机制的研究及其诊断、治疗等临床个体化方案的制定提供理论依据。