【作者】 王耀春；

【导师】 韩骅；

【作者基本信息】 第四军医大学 ， 发育生物学， 2009， 博士

【摘要】 单核细胞是机体先天性免疫和获得性免疫应答的重要组成部分,是单核吞噬系统的主要成员之一。大量研究表明骨髓单核细胞是树突状细胞和巨噬细胞的共同前体细胞,他们共同构成了外来微生物入侵时机体的有效防御系统,同时对于生理状态下清除机体内衰老和凋亡的细胞,维持机体的稳态发挥了不可替代的作用。树突状细胞是目前已知的功能最强大的抗原呈递细胞,是体内激发T细胞免疫应答的首要环节。而巨噬细胞能够吞噬细菌、病毒、凋亡的细胞及抗原抗体复合物等,并通过胞内溶酶体等将其消化清除,同时巨噬细胞还具有较强的抗原处理和递呈能力,能够通过分泌多种细胞因子发挥免疫调节和介导炎症反应。单核细胞,树突状细胞以及巨噬细胞的异常发育必然导致机体免疫系统功能和机体内环境的紊乱,最终引发机体的各种疾病。细胞的分化是一个复杂的生命过程,其中涉及多种信号途径的协同和调控。Notch信号途径在进化中高度保守,从线虫到人,从胚胎发育到成年个体的多种系统中都发挥着重要作用。Notch受体和配体家族均是单次跨膜蛋白,通过细胞间的相互作用,Notch受体胞内段发生水解入核,同时募集并结合多种蛋白分子形成转录共激活物,去除RBP-J的转录抑制作用,调控下游靶基因的表达。Notch信号途径对多细胞生物的很多不同组织中的细胞命运起着决定性作用,例如神经系统,血管系统,造血系统等等。在免疫系统的发育过程中,Notch信号途径在多个环节参与了免疫细胞发育命运的选择,如T/B细胞的分化、脾脏中边缘带B细胞和滤泡B细胞的分化等等。同时,Notch信号途径也是Th1和Th2免疫反应选择的调控因素之一。但Notch信号途径在其他免疫类型细胞诸如DC、巨噬细胞以及NK细胞发育的调控作用尚不十分清楚。虽然有报道显示阻断Notch信号途径可以抑制DC的发育,然而对于调控过程中的分子机制却是知之甚少。为了更加全面阐明Notch信号途径在免疫系统发育过程的调控作用,本课题利用条件性基因剔除技术,在小鼠造血干细胞中诱导剔除Notch信号途径的关键性转录因子RBP-J,在造血系细胞中彻底阻断Notch信号途径。通过RBP-J剔除后对单核细胞,DC和巨噬细胞发育及功能变化的深入探讨,进一步明确了Notch信号途径调控单核细胞命运选择中的作用,以及对DC和巨噬细胞功能的影响,并分析了相关的分子机制。主要研究成果如下:1.通过交配Mx-Cre转基因小鼠和RBP-Jflox/flox条件基因剔除小鼠,最终得到了同时携带Mx-Cre×RBP-Jflox/flox的双转基因条件剔除小鼠。经poly I-C诱导8-12周后得到RBP-J基因剔除小鼠。经Southern blot鉴定,骨髓中RBP-J剔除效率约接近100%。通过骨髓移植实验表明,剔除RBP-J使单核细胞在体内向DC方向的分化受到明显抑制,而向巨噬细胞方向的分化得到加强。体外的细胞诱导分化实验也印证了体内的实验结果。分子生物学检测表明RBP-J-/-巨噬细胞表达M-CSFR水平明显升高,提示Notch信号途径可能是通过直接或间接调控M-CSFR的表达调控单核细胞的分化方向。2.在DC中剔除RBP-J后,用LPS将不能有效刺激DC的发育成熟,DC发育停留在突起较少、MHCII表达较低的不成熟阶段,同时其迁移和抗原呈递功能受到损伤。进一步研究表明,趋化因子受体CXCR4是Notch信号途径下游的间接靶分子,在DC的发育过程中可以促进DC突起的长出和MHCII分子的表达。用慢病毒在RBP-J-/-DC中过表达CXCR4可以挽救由阻断Notch信号途径所引起的缺陷表型。我们的研究结果首次揭示了Notch信号可以通过调控CXCR4促进DC发育成熟。3.剔除RBP-J后,巨噬细胞在LPS刺激后不能向M1型巨噬细胞极化,而是被极化为M2型巨噬细胞,其迁移、吞噬、抗原加工以及呈递能力减退,失去了维持机体稳态的功能。在肿瘤中,RBP-J-/-巨噬细胞肿瘤杀伤功能减弱,肿瘤重量和体积没有因为巨噬细胞的存在受到抑制。免疫组化结果显示,RBP-J-/-巨噬细胞可以促进肿瘤中新生血管的生成,表明其具有肿瘤相关性巨噬细胞(TAM)的特性。同时,激活巨噬细胞中的Notch信号途径可以增强细胞的抗原呈递能力,并激发较强的Th1型免疫应答。这些结果表明Notch信号途径是巨噬细胞M1/M2极化的关键调控信号。综上所述,我们首次通过RBP-J条件性基因剔除小鼠,观察了Notch/RBP-J信号对于小鼠单核细胞来源DC和巨噬细胞发育和功能的调控作用。我们的结果表明,Notch信号参与调控单核细胞发育命运的方向;通过调控CXCR4的表达促进DC的成熟;同时对于巨噬细胞向M1方向极化是必须的。以上研究阐明了Notch信号途径在天然免疫系统发育中的重要作用,并充分解释了其作用机制,因此具有重要的理论和实际意义。更多还原

【Abstract】 Monocytes are the key components of innate and adaptive immunity, and are important in the mononuclear phagocytic system. Many studies have shown that bone marrow monocytes are common precursors for dendritic cells and macrophages, with build up the defense system effectively against the invasion of foreign micro-organisms. Meanwhile, these cells play an indispensable role in the removal of seniscent and apoptosis cells to maintain the homeostasis of bodies under physiological conditions. Dendritic cells are known to be the most powerful antigen -presenting cells and can activate T cells to initiate the immune responses in vivo. Macrophages, on the orther hand, can take up virus, bacteria, dead cells for apoptosis and antigen-antibody complex by phagocytosis and endocytosis, and remove them after degradation lysosomes. They also have strong antigen processing and presenting capacity and can play a variety of immune regulation roles and mediate inflammatory responses through secretion of cytokines. Abnormal development of monocytes, dendritic cells or macrophages will inevitably lead to disorders in the immune system and the internal environment, ultimately inducing various diseases of the body.Cell differetiation is a complex process, which requires a broad range of the crosstalk among signal transduction pathways. The Notch signaling pathway is highly conserved in evolution, and plays an important role in a variety of organisms from nematodes to humans, from embryonic development to adulthood. Notch receptor and ligand families belong to type I transmembrane proteins. When Notch receptor is triggered by direct interaction with its ligands, the intracellular domain of the Notch receptor (NICD) is released from the membrane after receptor cleavage executed by aγ-secretase-like protease. NICD translocates to nucleus and associates with RBP-J through its N-terminal RAM (RBP-J association molecule) domain, and transactivates promoters harboring RBP-J-binding sites, leading to the expression of genes associated cell differentiation. The Notch signaling pathway plays a decisive role in cell fate differentiation in many different systems, such as the nervous system, vascular system, hematopoietic system, etc. During the development of the immune system, the Notch signaling pathway is involved in various aspects of the differentiation of immune cells, including T versus B cell fate commitment and the marginal zone B versus follicular B cell fate commitment. Meanwhile, Notch signaling pathway can influence the differetiation of T cells in Th1 versus Th2 immune responses. However, the role of Notch signaling pathway in other types of cells such as DC, macrophages and NK cells is still not clear. Although there are reports that blocking Notch signaling can inhibit the development of DC, the molecular mechanism of the process has been poorly understood.In order to clarify the role of Notch signaling during the development of the immune system more clearly, using gene conditional knockout technology, we disrupted the key transcription factor RBP-J of Notch signaling pathway in mouse hematopoietic stem cells. In this study, we investigaed the changes of development and function of monocytes, DCs and macrophages after the deletion of RBP-J. The related molecular mechanisms are also analyzed using cellular and molecular technologies.The main finds are as follows:1. RBP-Jflox mice were crossed with Mx-Cre transgenic mice and to get Mx-Cre×RBP-Jflox/flox mice. We got RBP-J knockout mice after induction for 8-12 injecion of poly:I-C. Southern blot analysis of DNA isolated from bone marrow cells indicated about nearly 100% deletion efficiency.Bone marrow transplantation experiments showed that the differentiation of monocytes to DC was inhibited, but the differentiation to macrophages are strengthened upon RBP-J deletion. Cell differentiation by induction in vitro confirmed the experimental results in vivo. The expression of M-CSFR detected by molecular biology methods was significantly increased in RBP-J-/- macrophages, which indicated that the Notch signaling pathway might regulate the differentiation of monocytes through directly or indirectly controlling M-CSFR expression.2. We analyzed the changes of function and differentiation of DC after that RBP-J was removed. The results showed that LPS failed to stimulate DCs maturation, and DC development remained at an immature stage with low dendrites and low MHCII expression. At the same time their migration and antigen-presenting function were damaged. Further studies have shown that the chemokine receptor CXCR4 was an indirect downstream target of the Notch signaling pathway, which could promote out-growth of dendrites and the expression of MHCII during the development of DC phenotypic defects RBP-J-/- DC could be rescued by the over-expression of CXCR4 using lenti-virus. Our results showed that there was a signaling pathway that CXCR4 regulated by Notch could promote DC maturation.3. RBP-J-/- macrophages could not be polarized to the M1-type macrophages after LPS stimulation, but underwent for the M2-type differetiation. Their migration, phagocytosis, antigen processing and presentation abilities were all decreased. In tumors, RBP-J-/- macrophages had lower anti-tumor function, as shown by that tumor weight and size were suppressed by the existence of macrophages. Immunohistochemistry results showed that RBP-J-/- macrophages could promote tumor angiogenesis, indicating that it had certain characteristics of TAM. Meanwhile, activation of the Notch signaling pathway in macrophages could enhance their capacity of antigen-presenting and stimulate a strong Th1-type immune response. These results suggested that Notch signaling pathway should be activated in macrophages when these cells were used in treatment.In summary, we first provide the evidence that the development regulation of bone marrow monocyte-derived DC and macrophages was required by Notch/RBP-J signaling. Our results show that, Notch signals involved in regulating the fate decision of monocyte development, could promote the maturation of DC by regulating the expression of CXCR4, and was also necessary for the M1 macrophage polarization. Our results reveal understanding of the function and mechanisms of the Notch signaling pathway in the development of immune system.更多还原