【作者】 陶怡；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2005， 硕士

【摘要】 CD40是分子量为45—50kD的膜分子,属于TNFR超家族成员。主要表达在正常成熟B细胞上,参与多种生物学效应,如细胞增殖、分化、生发中心发育、Ig的分泌和同型转换、细胞表面分子CD80、CD86、CD95、ICAM-1的上调性表达和诱导细胞因子如IL-6、TNFα、LT和一些趋化因子的分泌。此外,CD40与EBV转化后的B细胞所表达的病毒抗原LMP1在信号传导方面有很多相似性。并且,当CD40配基化时,细胞表面CD40和LMP1分子可被募集到脂筏组成信号传导复合体传递信号。CD40的配体CD40L,又名CD154,是分子量为39kD的膜分子,属于TNF超家族成员。CD40L主要以三聚体的形式瞬时表达在抗原活化的CD4~+T细胞表面,也可以瞬时表达在生发中心的B细胞和异位表达在一些B细胞系来源的肿瘤细胞上。众所周知,B细胞的成熟需要通过在生发中心与抗原活化的、瞬时表达CD40L的CD4~+T细胞结合,促使B细胞表面的CD40配基化,才能使高抗原亲和力的、非自身免疫性的B细胞存活、增殖和分化。生发中心B-T细胞间CD40-CD40L的相互作用能够使B细胞免于凋亡。但是,生发中心中T细胞的数量很少,自身正常B细胞表达CD40L也是暂时的,这种严格的筛选机制只能使一部分功能性B细胞存活,而未能和T细胞结合发生配基化的恶性细胞或自身免疫性细胞则被清除。然而,许多肿瘤细胞能逃脱生发中心筛选机制,最终存活下来得到扩增,这种存活机制引起人们广泛兴趣。近年来,越来越多的文献报道受体和配体能共表达于同一细胞表面,尤其是肿瘤细胞上,这样自身可以形成信号传导的环路,而不需要外源因子的辅助。有可能一些致癌因素如病毒感染引起了这种异位表达或一些异位表达上调,从而参与了肿瘤细胞逃脱凋亡或免疫细胞攻击的机制。并且,这些受体和配体的共表达对肿瘤细胞具体作用如何,值得我们深入探讨。更多还原

【Abstract】 CD40, a 45-50kD membrane molecule, belongs to TNFR super-family. It is mainly expressed on mature B cells and can be involved in a variety of biological effects, such as cell proliferation, differentiation, germinal center development, Ig secretion and isotype switch , the up-regulation of cell molecules like CD80, CD86, CD95, ICAM-1 and the secretion of cytokines like IL-6, TNFα, LT and several chemokines. In addition, in the aspect of signal transduction, CD40 is similar to LMP1, one of the virus proteins expressed on B cells transformed by EBV. Furthermore, in the case of CD40 ligation, CD40 and LMP1 on the transformed cells can be recruited to lipid rafts to constitute a signalsome transducing signals. The ligand of CD40, CD40L, or CD154, a 39kD membrance molecule, belongs to TNF super family. CD40L is transiently expressed on antigen-activated CD4~+ T cells in the form of trimer. It can also be normally expressed on germinal center (GC) B cells or abnormally expressed on B cell-originated malignant cells. It is known to all that the maturation of B cells is dependent on the binding to CD40L transiently expressed on CD4~+ T cells within the GC, which promotes the ligation of CD40 on B cells and results in the survival, proliferation and differentiation of high-affinity, non-autorecreative B cells. CD40-CD40L interaction between B-T cells within the GC is able to refrain B cells from apoptosis. But, the number of T cells within the GC is limited and the expression of CD40L on GC B cells is transient. So, such a strict mechanism of selection leads to the survival of only a fraction of functional B cells, while the malignant or autoreactive B cells failing to bind to T cells are cleared out. However, many malignant cells can escape the selective mechanism within the GC and survive finally with a further proliferation, which draws great interests from people. In recentyears, more and more articles reported the fact that receptors and ligands could be co-expressed on the same cells, especially on malignant cells. In this way, the malignant cells can self-form autonomous signaling without external supports. It is possible that some carcinogenic factors like virus infection may result in the ectopic expressions or the up-regulation of such expressions, which can be involved in the mechanism of malignant cells escaping from apoptosis or the attack of immune cells. Furthermore, the exact effects of co-expression of receptors and ligands on malignant cells deserve us to explore.1. Effects of Epstein-Barr "Virus (EBV) infection on phenotype changes and biological characteristics of Multiple Myeloma (MM) cells Flow cytometry detection was carried out to analyze 10 cases of freshly isolated MM cells. The result showed the co-expression of CD40 and CD40L on No.2, 5, 9, 10 MM specimen cells, especially, the high co-expression on No.5, 10 specimens. Using EBV to infect MM cell line RPMI 8226 in vitro, we found that EBV could infect MM cells in vitro for the first time through the detection of LMP1 and EBNA-2 expression. EBV infection of RPMI8226 cells can up-regulate CD40 ligand on the level of both mRNA and protein, which could be involved in the anti-apoptosis function of the infected cells. In addition, the Confocal image showed two of CD40, CD40L and LMP1 molecules co-localized on infected cells. Furthermore, the chemokine receptor CXCR4 on RPMI8226 cells was up-regulated after EBV infection. And, in the presence of chemokine SDF-la, the ability of infected cells to migrate was improved. It is known to all, the ability to undergo anti-apoptosis and the ability to migrate are two important biological characteristics of malignant cells. In sum, it is possible that EBV, as one of the highly risky factors for MM, get involved in the origin and development of MM by transforming GC B cells to achieve anti-apoptosis function and by improving their ability to migrate from the GC to peripheral blood.2. The biological effects of co-expression of CD40 and CD40L on B lymphoma cells Two B lymphoma cell lines, Daudi and Raji, were chosen to make a further study. Flow cytometry detection revealed that CD40 and CD40 ligand were highly co-expressed on the two cell lines. Confocal microscope detection showed the co-localization of CD40 andCD40L on the membrane of Daudi or Raji cells. Antagnist antibody CD40mAb(3G3) or CD40LmAb(4Fl) established by our own institute was used to interdict CD40-CD40L signalling. Results showed that the antagonist antibody inhibited the growth, proliferation of lymphoma cells and induced their apoptosis in a dose-dependent manner with different cell lines. The conclusion was reached that the co-expression of CD40 and CD40L on Daudi or Raji cells contributed to the survival, growth, proliferation and the anti-apoptosis characteristics of malignant cells, providing a new target for the clinical application of immunotherapy to malignancies.更多还原