本文關(guān)鍵詞： 樹突狀細胞 E2F1 Gab2　出處：《中國科學(xué)技術(shù)大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：近來的研究發(fā)現(xiàn),轉(zhuǎn)錄因子E2F1除參與細胞周期調(diào)控,通過p53依賴和p53非依賴的兩種方式參與細胞凋亡外,在造血系統(tǒng)細胞的發(fā)育分化上還起到重要調(diào)節(jié)作用。E2F1基因敲除鼠中淋巴細胞顯著增生,但耐受下降并導(dǎo)致淋巴瘤。樹突狀細胞(DC)活化會誘導(dǎo)淋巴細胞增生,在E2F1基因敲除鼠中是否E2F1影響到DC的功能,從而引起淋巴細胞的異常?為此,本研究集中的研究了E2F1在樹突狀細胞中的調(diào)控作用和機制。 首先我們發(fā)現(xiàn),在LPS誘導(dǎo)的人外周血單核細胞來源樹突狀細胞和鼠源樹突狀細胞系DC2.4成熟過程中,E2F1的基因轉(zhuǎn)錄和蛋白表達都表現(xiàn)出瞬時下降。利用所建立的DC2.4敲低或高表達E2F1細胞為模型分析和發(fā)現(xiàn),敲低E2F1可導(dǎo)致DC2.4表型和功能都趨于成熟。相反,高表達E2F1能顯著抑制LPS誘導(dǎo)的DC2.4的成熟。通過E2F1基因敲除鼠骨髓來源的DC表型的檢測,驗證了上述的研究結(jié)果,提示了E2F1對DC成熟起到反饋調(diào)節(jié)作用。 為了進一步揭示E2F1調(diào)控樹突狀細胞成熟的分子機制,利用所建立的DC2.4基因修飾細胞模型分析發(fā)現(xiàn)：敲低E2F1導(dǎo)致包括Erk1/2, NF-κB和PI3K/Akt等涉及樹突狀細胞成熟的主要信號通路活性明顯增高。對E2F1可能參與的轉(zhuǎn)錄調(diào)節(jié)的部分基因分析發(fā)現(xiàn),E2F1可影響信號通路中重要的接頭蛋白Gab2的表達。對Gab2啟動子的分析和實驗研究發(fā)現(xiàn),E2F1可能通過與Spl相互作用,從而對Gab2轉(zhuǎn)錄調(diào)節(jié),并進一步影響了DC多個細胞信號通路的活性。 雖然Gab2參與多個細胞信號通路的活化,但Gab2的上調(diào)并未能完全模擬E2F1敲低所導(dǎo)致信號通路活性的所有變化,提示Gab2可能僅僅是眾多E2F1下游調(diào)控基因中的一個,而E2F1誘導(dǎo)DC成熟可能是多個因子共同作用的綜合結(jié)果。 本研究進一步擴展了轉(zhuǎn)錄因子E2F1的功能,首次闡明了其在DC成熟過程中的反饋調(diào)控作用及部分分子機制。研究結(jié)果顯示,在DC成熟過程中E2F1的表達經(jīng)歷了先下調(diào)后恢復(fù)的動態(tài)變化過程,因此推測E2F1的反饋調(diào)節(jié)作用主要是在DC細胞成熟后期,避免DC過度活化,起到了很好的平衡機體內(nèi)細胞穩(wěn)態(tài)的作用。雖然已有研究報道在腫瘤細胞中E2F1對Gab2的表達調(diào)控,但本研究中發(fā)現(xiàn)在DC中E2F1對Gab2轉(zhuǎn)錄調(diào)控利用了不同的機制。本研究提示,Gab2可能在免疫細胞中發(fā)育和分化的新功能。 樹突狀細胞的成熟和活化在免疫應(yīng)答中起到關(guān)鍵的作用,它功能的異常會直接關(guān)系到機體對病原體的抵抗和自身的保護。本研究成果為后續(xù)深入研究樹突狀細胞成熟的分子調(diào)控網(wǎng)絡(luò)及對治療用DC疫苗的研究打下了必要的基礎(chǔ)。
[Abstract]:Recent studies have found that transcription factor E2F1 participates in cell cycle regulation and apoptosis through p53 dependent and p53 independent approaches. It also plays an important role in regulating the development and differentiation of hematopoietic system cells. The lymphocytes proliferate significantly in E2F1 knockout mice. The activation of dendritic cells (DC) can induce the proliferation of lymphocytes, and whether E2F1 affects the function of DC in E2F1 knockout mice. And cause lymphocytic abnormalities? Therefore, this study focused on the regulatory role and mechanism of E2F1 in dendritic cells. First, we found that LPS induced human peripheral blood monocyte derived dendritic cells and murine dendritic cell line DC2.4 maturation process. The gene transcription and protein expression of E2F1 showed a transient decline. The established DC2.4 knockdown or overexpression E2F1 cells were used as model analysis and discovery. Knocking down E2F1 could lead to the maturation of phenotype and function of DC2.4. Overexpression of E2F1 could significantly inhibit the maturation of DC2.4 induced by LPS. The above results were verified by the detection of DC phenotypes from bone marrow of E2F1 knockout mice. These results suggest that E2F1 plays a feedback role in DC maturation. In order to further reveal the molecular mechanism of E2F1 regulating dendritic cell maturation, it was found that knockdown of E2F1 resulted in including Erk1/2 by using the established DC2.4 gene modified cell model. NF- 魏 B, PI3K/Akt and other major signaling pathways involved in dendritic cell maturation were significantly increased. Partial gene analysis of the transcriptional regulation involved in E2F1 was found. E2F1 could affect the expression of important junction protein Gab2 in signal pathway. The analysis and experimental study of Gab2 promoter showed that E2F1 might interact with Spl. Thus, Gab2 transcription was regulated and the activity of several signal pathways in DC cells was further affected. Although Gab2 is involved in the activation of multiple cell signaling pathways, the up-regulation of Gab2 does not fully mimic all the changes in signal pathway activity caused by E2F1 knockout. These results suggest that Gab2 may be only one of the many downstream regulatory genes of E2F1, and that E2F1 induces DC maturation may be a combined result of multiple factors. In this study, the function of transcription factor E2F1 was further expanded, and the feedback regulation and some molecular mechanisms of E2F1 during DC maturation were elucidated for the first time. During DC maturation, the expression of E2F1 experienced a dynamic process of down-regulation and then recovery. Therefore, it is assumed that the feedback regulation of E2F1 is mainly in the late stage of DC cell maturation to avoid the excessive activation of DC. E2F1 plays a good role in balancing the homeostasis of cells in the body, although it has been reported that E2F1 regulates the expression of Gab2 in tumor cells. In this study, we found that E2F1 used different mechanisms to regulate Gab2 transcription in DC. This study suggested that Gab2 2 might develop and differentiate in immune cells. The maturation and activation of dendritic cells play a key role in the immune response. The abnormality of its function is directly related to the organism's resistance to pathogens and its own protection. The results of this study have laid a solid foundation for the further study of the molecular regulatory network of dendritic cell maturation and the study of therapeutic DC vaccine. The basis of what you want.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392

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