本文選題：CD47 + CRL4��； 參考：《浙江大學》2017年碩士論文

【摘要】：CD47是一種在正常細胞上廣泛表達的蛋白,能作為"自我"的標志,'避免機體自身細胞被免疫細胞吞噬。但CD47通常在一些腫瘤細胞上會被過量地表達,導(dǎo)致這些腫瘤細胞能逃避免疫系統(tǒng)的吞噬。與CD47相對應(yīng)的受體SIRPα,是一種抑制性免疫受體,表達于髓系細胞。CD47-SIRPα中樞作為一個重要的免疫檢查點,在維持機體自身的穩(wěn)定以及對腫瘤細胞的清除方面起著重要的作用。研究表明,封閉CD47-SIRPα之間的相互作用能夠促進吞噬細胞(包括巨噬細胞和中性粒細胞)對腫瘤細胞的消滅。另外,靶向CD47-SIRPα中樞也可能會促進抗原呈遞細胞的功能從而刺激適應(yīng)性T細胞介導(dǎo)的抗腫瘤免疫反應(yīng)。因此,CD47-SIRP α中樞在腫瘤治療領(lǐng)域是一個非常有發(fā)展前景的先天性免疫檢查點。在本課題中,我們發(fā)現(xiàn)CD47能夠被Cu14A-DDB1E3泛素連接酶泛素化,進而被蛋白酶體降解。但當我們繼續(xù)探索CD47與DCAF之間的相互作用時,卻發(fā)現(xiàn)非常多的DCAF都能在免疫共沉淀實驗中與CD47發(fā)生相互作用。我們選擇了其中的DCAF3,DCAF5和DCAF8,在293T細胞系中進行了敲降實驗,我們發(fā)現(xiàn)敲降DCAF5時,CD47的水平有所上升。另外,我們利用CRISPR/Cas9技術(shù)在K562細胞系中對DCAF5,DCAF8和WSB1進行了敲除,并發(fā)現(xiàn)敲除DCAF5的情況下,CD47的水平也有所上升。我們進一步在B6小鼠體內(nèi)敲除了 DCAF5,我們發(fā)現(xiàn)DCAF5-/-小鼠骨髓細胞整體的CD47水平有所上升。根據(jù)以上結(jié)果,我們推測DCAF5有可能是介導(dǎo)CD47泛素化的DCAF。但要確證這一點,可能還需要更多的證據(jù)。總體來說,探索細胞表面CD47的調(diào)控方式是一個具有重要意義的問題,與腫瘤免疫治療領(lǐng)域的前沿密切相關(guān),值得我們進一步的研究。
[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 偽, a receptor corresponding to CD47, is an inhibitory immune receptor. It is expressed in myeloid cells. CD47-SIRP 偽, 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 偽 can promote the elimination of tumor cells by phagocytes (including macrophages and neutrophils). In addition, CD47-SIRP 偽 may promote the function of antigen-presenting cells and stimulate the anti-tumor immune response mediated by adaptive T cells. Therefore, CD47-SIRP 偽 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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.51
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本文編號：2054841