转录因子E2F1调节树突状细胞成熟的分子机制

发布时间：2018-01-21 19:23

本文关键词： 树突状细胞 E2F1 Gab2　出处：《中国科学技术大学》2012年博士论文　论文类型：学位论文

【摘要】：近来的研究发现,转录因子E2F1除参与细胞周期调控,通过p53依赖和p53非依赖的两种方式参与细胞凋亡外,在造血系统细胞的发育分化上还起到重要调节作用。E2F1基因敲除鼠中淋巴细胞显著增生,但耐受下降并导致淋巴瘤。树突状细胞(DC)活化会诱导淋巴细胞增生,在E2F1基因敲除鼠中是否E2F1影响到DC的功能,从而引起淋巴细胞的异常?为此,本研究集中的研究了E2F1在树突状细胞中的调控作用和机制。首先我们发现,在LPS诱导的人外周血单核细胞来源树突状细胞和鼠源树突状细胞系DC2.4成熟过程中,E2F1的基因转录和蛋白表达都表现出瞬时下降。利用所建立的DC2.4敲低或高表达E2F1细胞为模型分析和发现,敲低E2F1可导致DC2.4表型和功能都趋于成熟。相反,高表达E2F1能显著抑制LPS诱导的DC2.4的成熟。通过E2F1基因敲除鼠骨髓来源的DC表型的检测,验证了上述的研究结果,提示了E2F1对DC成熟起到反馈调节作用。为了进一步揭示E2F1调控树突状细胞成熟的分子机制,利用所建立的DC2.4基因修饰细胞模型分析发现：敲低E2F1导致包括Erk1/2, NF-κB和PI3K/Akt等涉及树突状细胞成熟的主要信号通路活性明显增高。对E2F1可能参与的转录调节的部分基因分析发现,E2F1可影响信号通路中重要的接头蛋白Gab2的表达。对Gab2启动子的分析和实验研究发现,E2F1可能通过与Spl相互作用,从而对Gab2转录调节,并进一步影响了DC多个细胞信号通路的活性。虽然Gab2参与多个细胞信号通路的活化,但Gab2的上调并未能完全模拟E2F1敲低所导致信号通路活性的所有变化,提示Gab2可能仅仅是众多E2F1下游调控基因中的一个,而E2F1诱导DC成熟可能是多个因子共同作用的综合结果。本研究进一步扩展了转录因子E2F1的功能,首次阐明了其在DC成熟过程中的反馈调控作用及部分分子机制。研究结果显示,在DC成熟过程中E2F1的表达经历了先下调后恢复的动态变化过程,因此推测E2F1的反馈调节作用主要是在DC细胞成熟后期,避免DC过度活化,起到了很好的平衡机体内细胞稳态的作用。虽然已有研究报道在肿瘤细胞中E2F1对Gab2的表达调控,但本研究中发现在DC中E2F1对Gab2转录调控利用了不同的机制。本研究提示,Gab2可能在免疫细胞中发育和分化的新功能。树突状细胞的成熟和活化在免疫应答中起到关键的作用,它功能的异常会直接关系到机体对病原体的抵抗和自身的保护。本研究成果为后续深入研究树突状细胞成熟的分子调控网络及对治疗用DC疫苗的研究打下了必要的基础。
[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.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392

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