Notch信号通路对小鼠巨噬细胞功能调控的研究
发布时间：2019-06-13 11:36
【摘要】： 巨噬细胞是重要的免疫细胞之一。巨噬细胞起源于骨髓造血干细胞,经髓系祖细胞、单核细胞分化而来。巨噬细胞可以通过清除凋亡细胞和产生生长因子的方式参与内环境稳态的维持,调控骨的形态发生、管腔分支形成、神经网络和新生血管形成。同时巨噬细胞活化是免疫应答的重要环节,可以通过吞噬杀伤作用和产生炎性因子发挥先天免疫作用,也可以通过抗原呈递功能启动T细胞和B细胞介导获得性免疫应答,其分泌的多种细胞因子对先天免疫和获得性免疫又具有调节作用。巨噬细胞功能异常与肿瘤和肥胖等多种疾病密切相关,因而深入研究巨噬细胞发育和功能的调控,对于进一步阐明稳态维持和免疫应答的发生机制,以及多种疾病的临床治疗都具有重要的意义。 Notch信号通路在进化上高度保守,通过局部细胞间的相互作用以控制细胞命运,是调控胚胎发育和多种成体组织器官体内稳态和细胞分化的重要信号通路。哺乳动物表达四个Notch受体(Notch1-4)和五个配体(Jagged1、Jagged2、和Delta-like-1、3、4)。细胞表面的Notch受体与配体结合后,在蛋白水解作用下释放胞内段(NICD)转位进入细胞核,与转录因子RBP-J相互作用,转而募集转录共激活物,活化下游靶基因的转录表达。 Notch信号通路是调控髓系造血细胞发育的重要途径,在造血干细胞的自我更新和髓系细胞的定向分化中都具有重要作用。同时Notch信号通路参与多种免疫细胞功能的调控,包括T细胞、B细胞和DC等。目前的研究提示,Notch信号通路对单核巨噬细胞的发育和功能具有重要的意义,但研究较少,而且结论不明确。因此,本课题同时应用条件性RBP-J基因剔除小鼠模型和巨噬细胞系RAW264.7稳定转染细胞模型,主要分析了Notch信号通路对巨噬细胞的增殖、抗原呈递功能和巨噬细胞活化的影响。主要研究结果如下: 1.获得了RBP-J条件性基因剔除小鼠,建立了小鼠巨噬细胞系RAW264.7的稳定转染Notch-1NICD和RBP-J(R218H)的细胞株。 通过对RBP-Jflox/wr和Mx-Cre转基因小鼠的繁育,获得了足够数量的的Mx-Cre-RBP-Jflox/wr和Mx-Cre-RBP-Jflox/flox转基因小鼠,给予腹腔注射12次poly I:C诱导Cre重组酶的表达后,成功获得了RBP-J基因剔除小鼠。应用脂质体法转染小鼠巨噬细胞系RAW264.7,经G418筛选和单克隆化,建立了稳定转染细胞株RAW-NIC和RAW-R218H,经q-PCR检测Notch通路下游基因HES-1表达水平证实,过表达Notch-1胞内段的RAW-NIC细胞可以有效激活Notch信号通路,而过表达RBP-J显性负突变蛋白RBP-J(R218H)的RAW-R218H细胞Notch信号通路抑制,成功获得了Notch信号通路活化和阻断的巨噬细胞系。RBP-J基因剔除小鼠和稳定转染细胞系的获得为后续研究建立了动物模型和细胞模型。 2. Notch信号通路阻断后分化成熟的小鼠腹腔巨噬细胞总数和比例无明显异常,Notch信号通路体外不影响巨噬细胞系的生长和增殖。 分离培养RBP-J剔除小鼠腹腔巨噬细胞,流式细胞术检测CD11b和F4/80双阳性细胞总数和比例,与对照小鼠相比,RBP-J剔除后小鼠腹腔巨噬细胞总数和比例都无明显差别;应用GSI体外阻断WT小鼠腹腔巨噬细胞的Notch通路后,腹腔巨噬细胞总数和比例也无明显异常。应用MTT法测定巨噬细胞稳定转染株的生长曲线,同时应用流式细胞术细胞分析周期,各组细胞均无明显差异。这些结果表明,Notch信号阻断并不影响小鼠腹腔巨噬细胞分化产生的总数和比例,Notch信号通路的体外活化或抑制并不影响巨噬细胞的生长和增殖。 3. Notch信号通路调控小鼠巨噬细胞的抗原呈递功能,这一作用可能是通过调控共刺激分子CD80和CD86的表达介导的。 小鼠腹腔巨噬细胞在RBP-J基因剔除或应用GSI体外阻断Notch信号通路后,体外与CFSE标记的初始T淋巴细胞共培养,流式细胞术分析表明增殖的T细胞总数和比例显著低于对照组。进一步研究发现Notch信号阻断后腹腔巨噬细胞膜表面共刺激分子CD80和CD86表达显著减低。而且,巨噬细胞系Notch信号通路活化后,可以促进更多的T细胞增殖。这些结果表明,Notch信号通路可能通过调控小鼠巨噬细胞膜表面共刺激分子CD80和CD86的表达,调控巨噬细胞的抗原呈递功能。 4. Notch信号通路阻断后,LPS不能诱导小鼠巨噬细胞的经典活化,Notch信号参与调控巨噬细胞对LPS应答中多种炎性细胞因子的产生。 Notch信号通路参与LPS诱导的巨噬细胞活化,q-PCR证实LPS活化后,巨噬细胞多个Notch受体和配体的表达改变。RBP-J基因剔除或应用GSI体外阻断Notch信号通路后,小鼠腹腔巨噬细胞对LPS的应答改变,与对照相比,IL-12产生显著减少,而IL-10产生轻度增加,不能活化为M1型巨噬细胞,而接近M2巨噬细胞的表型。此外,Notch信号通路可以促进LPS诱导的巨噬细胞产生TNF-α和iNOS,抑制IL-1β的产生,阻断Notch信号后IL-6的产生增多。这些结果表明,LPS诱导巨噬细胞经典活化过程中依赖Notch通路的作用,Notch信号的缺失导致巨噬细胞M1型活化障碍,而Notch信号通路与LPS共同调控巨噬细胞多种炎性因子的产生。 5. Notch信号通路调控巨噬细胞CD11b的表达。 通过剔除RBP-J或体外应用GSI阻断小鼠腹腔巨噬细胞的Notch信号通路,巨噬细胞表达的CD11b在蛋白水平和mRNA水平均显著减低;活化巨噬细胞系的Notch信号通路可以上调CD11bmRNA水平和蛋白水平的表达。进一步将小鼠CD11b启动子区克隆构建基因报告质粒,通过报告基因实验证实,Notch信号通路对CD11b基因的转录无直接调控作用,可能通过间接作用调控CD11b的表达。 结论 通过本课题的研究证实,Notch信号通路可以调控巨噬细胞的多种生物学特征。巨噬细胞发育过程中,Notch信号通路的阻断不影响小鼠腹腔巨噬细胞分化产生的细胞总数和比例,体外实验中Notch信号通路不调控巨噬细胞的生长和增殖。然而,Notch信号通路可以调控小鼠巨噬细胞的抗原呈递功能,这一作用可能是通过调控共刺激分子CD80和CD86的表达介导的。LPS诱导巨噬细胞经典活化过程中依赖Notch通路的作用,Notch信号的缺失导致巨噬细胞M1型活化障碍,而且Notch信号通路与LPS共同调控巨噬细胞多种炎性因子的产生。此外,Notch信号通路还可能通过间接作用调控巨噬细胞膜表面重要标记分子CD11b的表达,这一作用可能成为Notch信号途径参与调控巨噬细胞发育和功能的另一靶点。
[Abstract]:Macrophages are one of the most important immune cells. Macrophages originate from the bone marrow hematopoietic stem cells and are differentiated from the myeloid progenitors and monocytes. Macrophages can be involved in the maintenance of homeostasis of the internal environment by removing the apoptotic cells and the production of growth factors, regulating the morphogenesis of the bone, the formation of the lumen branches, the neural network and the formation of new blood vessels. At the same time, the activation of the macrophage is an important part of the immune response, and can play a innate immune function through the phagocytosis of the killing effect and the production of the inflammatory factors, and the T-cell and the B-cell-mediated acquired immune response can be started by the antigen-presenting function, The secretion of various cytokines has a regulating effect on the innate immunity and the acquired immunity. The function of macrophage is closely related to many diseases, such as tumor and obesity. Therefore, the regulation of macrophage development and function is deeply studied, and it is of great significance for further elucidating the mechanism of steady-state maintenance and immune response, as well as the clinical treatment of various diseases. The Notch signaling pathway is highly conserved in evolution, and the cell fate is controlled by the interaction between the local cells. It is an important letter to regulate the steady-state and cellular differentiation of the embryonic development and various adult tissue organs. Route. Mammals express four Notch receptors (Notch1-4) and five ligands (Jagged1, Jagged2, and Delta-like-1,3 And (4), after the Notch receptor on the surface of the cell is combined with the ligand, releasing the intracellular segment (NICD) into the cell nucleus under the action of protein hydrolysis, and interacting with the transcription factor RBP-J, and in turn, raising the transcription co-activator, and activating the rotation of the downstream target gene. The Notch signaling pathway is an important way to regulate the development of myeloid hematopoietic cells, and in the self-renewal of hematopoietic stem cells and the directional differentiation of myeloid cells The Notch signaling pathway is involved in the regulation of various immune cell functions, including T cells, The present study suggests that the Notch signaling pathway plays an important role in the development and function of mononuclear macrophages, but the study is less, In this study, the cell model of the mouse model and the macrophage-based RAW264.7 was transfected with the conditional RBP-J gene at the same time, and the proliferation, the antigen-presenting function and the macrophagocytosis of the Notch signaling pathway to the macrophages were mainly analyzed. The effect of cell activation. The results of this study were as follows:1. The RBP-J conditioned gene was obtained, and the stable transfection of Notch-1 NICD and RBP-J in the mouse macrophage system RAW264.7 was established. (R218H), a sufficient number of Mx-Cre-RBP-Jfloor/ wr and Mx-Cre-RBP-Jfloor/ flox transgenic mice were obtained by the propagation of the RBP-Jfloating/ wr and Mx-Cre transgenic mice, and the expression of the Cre recombinase was induced by intraperitoneal injection of 12 polyI: C. The RBP-J gene knockout mice were obtained by using the liposome method, and the mouse macrophage system RAW264.7 was transfected with the liposome method, and the stably transfected cell line RAW-NIC and RAW-R218H were established by G418, and the expression level of the HES-1 downstream of the Notch pathway was confirmed by the q-PCR, and the RAW-NIC cells of the Notch-1 intracellular segment were overexpressed. The Notch signaling pathway can be effectively activated, and the Notch signaling pathway in the RAW-R218H cell of the overexpressing RBP-J dominant negative mutant protein RBP-J (R218H) is inhibited, and Notch is successfully obtained. Activated and blocked macrophage system of signal pathway. RBP-J gene knockout mouse and stably transfected cell line The animal model and cell model were established in the study.2. The total number and proportion of peritoneal macrophages in the mature mice after the blockade of the Notch signaling pathway were not significantly abnormal, and the Notch signaling The number and proportion of CD11b and F4/80 double-positive cells were detected by flow cytometry, compared with control mice. There was no significant difference in the total number and proportion of the peritoneal macrophages of the mice after J-removal. The total number and proportion of macrophages in the peritoneal macrophages were not significantly abnormal after the ch pathway. The growth curve of the stable transfected cells was determined by MTT method, and at the same time, the growth curve of the stable transfected cells was determined by MTT method. The results showed that Notch signaling could not affect the total number and proportion of mouse peritoneal macrophage differentiation and Notch signaling. The in vitro activation or inhibition of the pathway does not affect the growth and proliferation of macrophages. The expression of the co-stimulatory molecules CD80 and CD86 may be mediated by the regulation of the expression of the co-stimulatory molecules CD80 and CD86. The results of co-culture and flow cytometry showed that the total number and proportion of T cells were significantly lower in the control group than in the control group. The expression of CD80 and CD86 on the membrane surface of the peritoneal macrophages was significantly reduced. After activation of the Notch signaling pathway in the macrophage system, more T-cell proliferation can be promoted. These results suggest that the Notch signaling pathway may be regulated by modulating the surface of the mouse macrophage membrane The expression of the co-stimulatory molecules CD80 and CD86 can regulate the antigen-presenting function of the macrophages. The Notch signaling is involved in the regulation of the production of a variety of inflammatory cytokines in the LPS response. The Notch signaling pathway is involved in LPS-induced macrophagocytosis Activation of the cell and q-PCR confirmed the change of expression of a plurality of Notch receptors and ligands in the macrophage after LPS activation. The response of the mouse peritoneal macrophages to the LPS was changed after the RBP-J gene was removed or the Notch signaling pathway was blocked in vitro by the GSI, and the IL-12 production was significantly reduced as compared to the control. In addition, the Notch signaling pathway can promote the generation of TN by LPS-induced macrophages. F-1 and iNOS inhibited the production of IL-1 and the increase of IL-6 after the blocking of the Notch signal. The results showed that the role of the Notch pathway in the classical activation of macrophages induced by LPS and the absence of Notch signaling resulted in the macrophages. M1 activation disorder, and Notch signaling pathway is co-regulated with LPS The expression of the macrophage CD11b was regulated by the Notch signaling pathway. The expression of CD11b in the peritoneal macrophages of the mouse was blocked by the elimination of RBP-J or in vitro. The expression of CD11b in the macrophages was at the level of the protein and the mRNA water. The expression of the expression of CD11b mRNA and the level of the protein can be up-regulated by the Notch signaling pathway of the activated macrophage system. ,N The Tch signal pathway has no direct regulatory effect on the transcription of the CD11b gene and may be regulated indirectly by indirect action. The results of this study confirm that the Notch signaling pathway can regulate the various biological characteristics of macrophages. In the process of macrophage development, Notch signaling The blocking of the pathway does not affect the total number and proportion of the cells produced by the differentiation of the peritoneal macrophages of the mouse. In vitro, the Notch signaling pathway does not regulate the growth and proliferation of the macrophages. However, Not The ch signal pathway can regulate the antigen-presenting function of the mouse macrophages, which may be mediated by the regulation of the expression of the co-stimulatory molecules CD80 and CD86. The role of the Notch pathway in the classical activation of the macrophages induced by LPS, N The absence of the och signal leads to the activation of the macrophage M1, and the Notch signaling pathway is co-regulated with the LPS to control the production of a variety of inflammatory factors. In addition, the Notch signaling pathway may also regulate the autophagy indirectly by indirect action.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
本文编号：2498505
[Abstract]:Macrophages are one of the most important immune cells. Macrophages originate from the bone marrow hematopoietic stem cells and are differentiated from the myeloid progenitors and monocytes. Macrophages can be involved in the maintenance of homeostasis of the internal environment by removing the apoptotic cells and the production of growth factors, regulating the morphogenesis of the bone, the formation of the lumen branches, the neural network and the formation of new blood vessels. At the same time, the activation of the macrophage is an important part of the immune response, and can play a innate immune function through the phagocytosis of the killing effect and the production of the inflammatory factors, and the T-cell and the B-cell-mediated acquired immune response can be started by the antigen-presenting function, The secretion of various cytokines has a regulating effect on the innate immunity and the acquired immunity. The function of macrophage is closely related to many diseases, such as tumor and obesity. Therefore, the regulation of macrophage development and function is deeply studied, and it is of great significance for further elucidating the mechanism of steady-state maintenance and immune response, as well as the clinical treatment of various diseases. The Notch signaling pathway is highly conserved in evolution, and the cell fate is controlled by the interaction between the local cells. It is an important letter to regulate the steady-state and cellular differentiation of the embryonic development and various adult tissue organs. Route. Mammals express four Notch receptors (Notch1-4) and five ligands (Jagged1, Jagged2, and Delta-like-1,3 And (4), after the Notch receptor on the surface of the cell is combined with the ligand, releasing the intracellular segment (NICD) into the cell nucleus under the action of protein hydrolysis, and interacting with the transcription factor RBP-J, and in turn, raising the transcription co-activator, and activating the rotation of the downstream target gene. The Notch signaling pathway is an important way to regulate the development of myeloid hematopoietic cells, and in the self-renewal of hematopoietic stem cells and the directional differentiation of myeloid cells The Notch signaling pathway is involved in the regulation of various immune cell functions, including T cells, The present study suggests that the Notch signaling pathway plays an important role in the development and function of mononuclear macrophages, but the study is less, In this study, the cell model of the mouse model and the macrophage-based RAW264.7 was transfected with the conditional RBP-J gene at the same time, and the proliferation, the antigen-presenting function and the macrophagocytosis of the Notch signaling pathway to the macrophages were mainly analyzed. The effect of cell activation. The results of this study were as follows:1. The RBP-J conditioned gene was obtained, and the stable transfection of Notch-1 NICD and RBP-J in the mouse macrophage system RAW264.7 was established. (R218H), a sufficient number of Mx-Cre-RBP-Jfloor/ wr and Mx-Cre-RBP-Jfloor/ flox transgenic mice were obtained by the propagation of the RBP-Jfloating/ wr and Mx-Cre transgenic mice, and the expression of the Cre recombinase was induced by intraperitoneal injection of 12 polyI: C. The RBP-J gene knockout mice were obtained by using the liposome method, and the mouse macrophage system RAW264.7 was transfected with the liposome method, and the stably transfected cell line RAW-NIC and RAW-R218H were established by G418, and the expression level of the HES-1 downstream of the Notch pathway was confirmed by the q-PCR, and the RAW-NIC cells of the Notch-1 intracellular segment were overexpressed. The Notch signaling pathway can be effectively activated, and the Notch signaling pathway in the RAW-R218H cell of the overexpressing RBP-J dominant negative mutant protein RBP-J (R218H) is inhibited, and Notch is successfully obtained. Activated and blocked macrophage system of signal pathway. RBP-J gene knockout mouse and stably transfected cell line The animal model and cell model were established in the study.2. The total number and proportion of peritoneal macrophages in the mature mice after the blockade of the Notch signaling pathway were not significantly abnormal, and the Notch signaling The number and proportion of CD11b and F4/80 double-positive cells were detected by flow cytometry, compared with control mice. There was no significant difference in the total number and proportion of the peritoneal macrophages of the mice after J-removal. The total number and proportion of macrophages in the peritoneal macrophages were not significantly abnormal after the ch pathway. The growth curve of the stable transfected cells was determined by MTT method, and at the same time, the growth curve of the stable transfected cells was determined by MTT method. The results showed that Notch signaling could not affect the total number and proportion of mouse peritoneal macrophage differentiation and Notch signaling. The in vitro activation or inhibition of the pathway does not affect the growth and proliferation of macrophages. The expression of the co-stimulatory molecules CD80 and CD86 may be mediated by the regulation of the expression of the co-stimulatory molecules CD80 and CD86. The results of co-culture and flow cytometry showed that the total number and proportion of T cells were significantly lower in the control group than in the control group. The expression of CD80 and CD86 on the membrane surface of the peritoneal macrophages was significantly reduced. After activation of the Notch signaling pathway in the macrophage system, more T-cell proliferation can be promoted. These results suggest that the Notch signaling pathway may be regulated by modulating the surface of the mouse macrophage membrane The expression of the co-stimulatory molecules CD80 and CD86 can regulate the antigen-presenting function of the macrophages. The Notch signaling is involved in the regulation of the production of a variety of inflammatory cytokines in the LPS response. The Notch signaling pathway is involved in LPS-induced macrophagocytosis Activation of the cell and q-PCR confirmed the change of expression of a plurality of Notch receptors and ligands in the macrophage after LPS activation. The response of the mouse peritoneal macrophages to the LPS was changed after the RBP-J gene was removed or the Notch signaling pathway was blocked in vitro by the GSI, and the IL-12 production was significantly reduced as compared to the control. In addition, the Notch signaling pathway can promote the generation of TN by LPS-induced macrophages. F-1 and iNOS inhibited the production of IL-1 and the increase of IL-6 after the blocking of the Notch signal. The results showed that the role of the Notch pathway in the classical activation of macrophages induced by LPS and the absence of Notch signaling resulted in the macrophages. M1 activation disorder, and Notch signaling pathway is co-regulated with LPS The expression of the macrophage CD11b was regulated by the Notch signaling pathway. The expression of CD11b in the peritoneal macrophages of the mouse was blocked by the elimination of RBP-J or in vitro. The expression of CD11b in the macrophages was at the level of the protein and the mRNA water. The expression of the expression of CD11b mRNA and the level of the protein can be up-regulated by the Notch signaling pathway of the activated macrophage system. ,N The Tch signal pathway has no direct regulatory effect on the transcription of the CD11b gene and may be regulated indirectly by indirect action. The results of this study confirm that the Notch signaling pathway can regulate the various biological characteristics of macrophages. In the process of macrophage development, Notch signaling The blocking of the pathway does not affect the total number and proportion of the cells produced by the differentiation of the peritoneal macrophages of the mouse. In vitro, the Notch signaling pathway does not regulate the growth and proliferation of the macrophages. However, Not The ch signal pathway can regulate the antigen-presenting function of the mouse macrophages, which may be mediated by the regulation of the expression of the co-stimulatory molecules CD80 and CD86. The role of the Notch pathway in the classical activation of the macrophages induced by LPS, N The absence of the och signal leads to the activation of the macrophage M1, and the Notch signaling pathway is co-regulated with the LPS to control the production of a variety of inflammatory factors. In addition, the Notch signaling pathway may also regulate the autophagy indirectly by indirect action.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
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