CRL4 E3泛素连接酶调控CD47分子机制的研究

发布时间：2018-06-23 00:12

  本文选题：CD47 + CRL4　； 参考：《浙江大学》2017年硕士论文

【摘要】：CD47是一种在正常细胞上广泛表达的蛋白,能作为"自我"的标志,'避免机体自身细胞被免疫细胞吞噬。但CD47通常在一些肿瘤细胞上会被过量地表达,导致这些肿瘤细胞能逃避免疫系统的吞噬。与CD47相对应的受体SIRPα,是一种抑制性免疫受体,表达于髓系细胞。CD47-SIRPα中枢作为一个重要的免疫检查点,在维持机体自身的稳定以及对肿瘤细胞的清除方面起着重要的作用。研究表明,封闭CD47-SIRPα之间的相互作用能够促进吞噬细胞(包括巨噬细胞和中性粒细胞)对肿瘤细胞的消灭。另外,靶向CD47-SIRPα中枢也可能会促进抗原呈递细胞的功能从而刺激适应性T细胞介导的抗肿瘤免疫反应。因此,CD47-SIRP α中枢在肿瘤治疗领域是一个非常有发展前景的先天性免疫检查点。在本课题中,我们发现CD47能够被Cu14A-DDB1E3泛素连接酶泛素化,进而被蛋白酶体降解。但当我们继续探索CD47与DCAF之间的相互作用时,却发现非常多的DCAF都能在免疫共沉淀实验中与CD47发生相互作用。我们选择了其中的DCAF3,DCAF5和DCAF8,在293T细胞系中进行了敲降实验,我们发现敲降DCAF5时,CD47的水平有所上升。另外,我们利用CRISPR/Cas9技术在K562细胞系中对DCAF5,DCAF8和WSB1进行了敲除,并发现敲除DCAF5的情况下,CD47的水平也有所上升。我们进一步在B6小鼠体内敲除了 DCAF5,我们发现DCAF5-/-小鼠骨髓细胞整体的CD47水平有所上升。根据以上结果,我们推测DCAF5有可能是介导CD47泛素化的DCAF。但要确证这一点,可能还需要更多的证据。总体来说,探索细胞表面CD47的调控方式是一个具有重要意义的问题,与肿瘤免疫治疗领域的前沿密切相关,值得我们进一步的研究。
[Abstract]:CD47, a protein widely expressed in normal cells, acts as a "self" marker to prevent the body's own cells from being swallowed up by immune cells. But CD47 is often overexpressed in some tumor cells, causing them to escape the immune system's phagocytosis. SIRP 伪, a receptor corresponding to CD47, is an inhibitory immune receptor. It is expressed in myeloid cells. CD47-SIRP 伪, as an important immunological checkpoint, plays an important role in maintaining the stability of the body and clearing the tumor cells. It has been shown that blocking the interaction between CD47-SIRP 伪 can promote the elimination of tumor cells by phagocytes (including macrophages and neutrophils). In addition, CD47-SIRP 伪 may promote the function of antigen-presenting cells and stimulate the anti-tumor immune response mediated by adaptive T cells. Therefore, CD47-SIRP 伪 center is a promising congenital immunological checkpoint in the field of tumor therapy. In this study, we found that CD47 could be decomposed by Cu14A-DDB1E3 ubiquitin ligase, and then degraded by proteasome. However, when we continue to explore the interaction between CD47 and DCAF, we find that many DCAF can interact with CD47 in immunoprecipitation. We selected DCAF3, DCAF5 and DCAF8, and carried out knock-down experiments in 293T cell line. We found that the level of CD47 increased when DCAF5 was knocked down. In addition, we used CRISPRR / Cas9 technique to knock out DCAF5, DCAF8 and WSB1 in K562 cell line, and found that the level of CD47 also increased with the knockout of DCAF5. We further knocked out DCAF5 in B6 mice, and we found that CD47 levels in bone marrow cells as a whole increased in DCAF5-r-mice. Based on the above results, we speculate that DCAF5 may be a CD47 ubiquitin mediated DCAF5. But more evidence may be needed to confirm this. In general, exploring the regulation of CD47 on cell surface is an important issue, which is closely related to the frontier of tumor immunotherapy, and is worthy of further study.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.51
，

本文编号：2054841