【作者】 赵向峰；

【导师】 朱迅；

【作者基本信息】 吉林大学 ， 免疫学， 2010， 博士

【摘要】 巨噬细胞是一类广泛存在于组织中的单核吞噬细胞,它是机体内天然免疫的第一道屏障。同时,巨噬细胞在免疫监视中也发挥重要作用。巨噬细胞可以通过分泌如TNF、NO等杀伤性效应分子来杀伤肿瘤细胞;另一方面,巨噬细胞可以通过细胞间接触来直接杀伤肿瘤细胞,而对于巨噬细胞的直接杀伤活性的机制研究一直是肿瘤免疫的一个重要领域。通过对已经鉴定出来的细胞膜表面杀伤效应分子的研究发现,封闭这些效应分子后巨噬细胞仍然具有很强的肿瘤杀伤活性,这一现象提示说明巨噬细胞膜表面存在一些尚未为人所知的分子,这些分子在巨噬细胞杀伤肿瘤的进程中起关键作用。本实验室前期研究发现,BCG刺激活化的巨噬细胞可以杀伤MCA207肿瘤细胞,这一过程与其细胞膜表面特有的蛋白分子密切相关,通过SDS-PAGE联合质谱检测的方法对BCG刺激活化和TGC诱导活化的巨噬细胞膜蛋白进行比对,我们得到了454个在BCG刺激活化的巨噬细胞膜表面上调表达的蛋白。为了揭示这些分子在巨噬细胞杀伤肿瘤过程中的作用,我们从中选取了两个新蛋白NMAAP1和Trim59进行基因克隆及其功能研究。第一部分:首先对本实验室的前期成果进行了总结,并利用生物信息学的手段从中筛选出了两个待研究的新蛋白,其名称分别是NMAAP1和Trim59,并对这两个蛋白的潜在功能做了预测。结果:NMAAP1可以与DAPK结合,进而调控细胞凋亡的进程;另一方面,NMAAP1的同源家族成员kiaa1754可以与IP3R结合,进而调控钙离子通道,这一进程可能参与细胞分化;同时NMAAP1蛋白含有一个Mab-21结构域,在生物进化、系统发育等多方面都有这一结构的身影。另一个新蛋白Trim59的预测结果表明,这一蛋白含有几个可能参与分子间相互作用的结构,分别是ring finger、B-box以及卷曲螺旋,蛋白功能预测结果显示,Trim59可能与细胞间接触密切相关。第二部分:克隆并表达了这两个新蛋白,通过亲和层析的方法得到了两种纯化后的融合蛋白。然后利用这两个纯化后的蛋白作为抗原制备了多克隆抗体。利用Western-blotting检测了这两种抗体的特异性后,我们进行了免疫组化实验,对这两个蛋白在小鼠体内不同组织的分布进行了鉴定。结果:通过原核表达的方式,得到了两个融合蛋白,并利用其制备了多克隆抗体,SDS-PAGE电泳以及western-blotting结果表明所纯化的两个蛋白正是我们所需要的目的蛋白,同时也证明了我们的抗体具有很强的特异性。通过免疫组化,证明了我们的抗体可以和天然构象下的蛋白正常结合。同时也分析了两个蛋白在机体内不同组织的分布情况,结果显示:NMAAP1在胸腺组织中的表达量较高,这可能与IP3R可以调节TCR敏感性进而参与T细胞的活化相关。Trim59在脾脏组织中的表达量较高,提示Trim59可能在免疫系统中扮演重要角色;另外,在卵巢组织中也检测到大量的Trim59表达,提示Trim59可能在生殖系统中也有作用。第三部分:利用自制的多克隆抗体进行了抗体封闭细胞毒实验,对我们所选取的两个新蛋白在巨噬细胞杀伤肿瘤过程中所发挥的作用进行了初步研究。另外,我们克隆了Trim59的全长基因,并将其稳定转染于Raw264.7细胞,通过对转染前后的Raw264.7细胞生物学性状的研究来鉴定Trim59的具体功能。结果:通过抗体封闭细胞毒实验发现,我们所选取的蛋白Trim59在BCG刺激活化的巨噬细胞杀伤肿瘤的过程中起关键作用,而NMAAP1则在杀伤过程中几乎没有作用。为了进一步研究Trim59在杀伤过程中所扮演的角色,我们将Trim59转染后表达于Raw264.7细胞系,并检测了这一细胞的肿瘤杀伤活性。实验结果表明,Trim59并不直接参与杀伤。通过对转染后细胞的吞噬活力进行检测,发现Trim59可以促进巨噬细胞对异物的吞噬,这可能与Trim59可以促进巨噬细胞与其它分子之间相互作用有关。因而我们推断Trim59在巨噬细胞杀伤的过程中是一个辅助分子,它可以通过促进巨噬细胞与靶细胞接触来加强巨噬细胞的细胞毒作用。综上所述,NMAAP1在胸腺组织中表达量较高,但其在巨噬细胞直接杀伤肿瘤的过程中没有明显作用,结合它的分子结构我们预测,NMAAP1可能参与巨噬细胞的特异性分化。Trim59在脾脏及卵巢中的表达量很高,提示其可能与机体免疫及生殖相关;Trim59是巨噬细胞杀伤肿瘤过程中的关键效应分子,但它是以一个辅助效应分子的身份在起作用,Trim59本身可以促进巨噬细胞对异物的吞噬作用。更多还原

【Abstract】 Macrophage is one of mononuclear cells in the body which widely exist in organizations; it is the first barrier of natural immune system. Macrophage also plays an important role in immune monitoring. Macrophages can secrete such as TNF, NO to kill cancer cells, on the other hand, macrophage could kill tumor cells directly through cell to cell contact,. The mechanism of this process is an important territory of tumor immunity. Up to date, lots of studies show that there must be some novel molecules on the membrane of macrophages, which mediate the cytotoxic process against tumor cells.In previous study, we found that BCG activated macrophages could kill MCA207 tumor cells through cell to cell contact. Membrane proteins from BCG activated macrophages and thioglycolate elicited macrophages were compared with a new method of SDS-page combine liquid chromatographytandem mass spectrometry. Comparisons resulted in a list of 454 proteins which were identified from BCG activated microphages only. From these proteins, we selected two novel proteins as our research targets.Part 1:We selected two novel proteins from our previous study using bioinformatics, named NMAAP1 and Trim59, and predicted the potential function of these two novel proteins.Result: NMAAP1 could combine with DAPK to mediate cell apoptosis; on the other hand, kiaa1754 (the homology member of NMAAP1) could combine to IP3R to mediate the Calcium Ion Channels, which might be the most important process in cell differentiation; meanwhile, NMAAP1 have a structure named Mab-21, which possibly mediate the organic evolution and phylogenetic development. Trim59 have several structures which might be involved in cell to cell contact.Part 2:We cloned and expressed the two proteins, and purified the proteins by affinity chromatography. Using these two proteins, we generated two polyclonal antibodies against these two proteins. After western-blotting detection, immunohistochemisty was processed.Result: In this study, we cloned the two novel proteins to generated fusion proteins in E. coil. The purified fusion proteins were applied for generation of polyclonal antibodies. Western-blotting detection showed that the polyclonal antibodies have high specificities to recognize target proteins. Using of the polyclonal antibodies, we detected the tissue distribution of these two novel proteins, and this process also showed our antibodies could combine to the nature protein. The results showed that NMAAP1 abundantly expressed in the thymus, which might be involved in T cell differentiation through IP3R combine to TCR. Trim59 abundantly expressed in the spleen, which indicate Trim59 is an important molecule in the immune system; on the other hand, Trim59 was detected from ovary, which indicate Trim59 might be involved in reproductive system.Part 3:We detected functions of the two novel proteins through antibodies blocked cell cytotoxicity. We cloned the full length of Trim59 and stably transfected it into Raw264.7 cell lines to investigate the function of Trim59.Result: Using the antibodies to block Trim59 on the membrane of BCG activated macrophages significantly reduced BCG activated macrophages’cytotoxicity against MCA207 tumor cells. This potentiated that Trim59 serves as an indispensable molecule in maintaining BCG activated macrophages’activity. However, blocking of NMAAP1 failed to down-regulate the cytotoxicity of BCG activated macrophages against MCA207 tumor cells. This result indicated that NMAAP1 is not the necessary molecule in BCG activated macrophages tumoricidal process.Overexpression of Trim59 in Raw264.7 cell line failed to lyse target MCA207 cells, which demonstrated Trim59 per se could not enhance macrophages cytotoxicity. Our results imply Trim59 might be an essential accessory molecule in mediating BCG activated macrophages tumoricidal functions. Interestingly, the phagocytosis of transfected Raw264.7 cells was enhanced notably; this might be related to the structure of Trim59, which have a ring finger domain, a B-box domain, and two coli-coli domains. We infer that Trim59 is an associated molecule, which promote macrophages to get in touch with tumor cells.Conclusion, NMAAP1 has a high expression in the thymus, and is not the necessary molecule in macrophages directly tumoricidal process, the structure of NMAAP1 indicate it might involved in the activate process. Trim59 has a high expression in spleen and ovary, which indicate Trim59 involved in immunization and reproduction. Trim59 is an indispensably molecule in macrophages tumoricidal process, and Trim59 act as an associated molecule in this process. On another hand, Trim59 could enhance the phagocytosis of macrophages.更多还原