【摘要】： 巨噬細(xì)胞是重要的免疫細(xì)胞之一。巨噬細(xì)胞起源于骨髓造血干細(xì)胞,經(jīng)髓系祖細(xì)胞、單核細(xì)胞分化而來。巨噬細(xì)胞可以通過清除凋亡細(xì)胞和產(chǎn)生生長(zhǎng)因子的方式參與內(nèi)環(huán)境穩(wěn)態(tài)的維持,調(diào)控骨的形態(tài)發(fā)生、管腔分支形成、神經(jīng)網(wǎng)絡(luò)和新生血管形成。同時(shí)巨噬細(xì)胞活化是免疫應(yīng)答的重要環(huán)節(jié),可以通過吞噬殺傷作用和產(chǎn)生炎性因子發(fā)揮先天免疫作用,也可以通過抗原呈遞功能啟動(dòng)T細(xì)胞和B細(xì)胞介導(dǎo)獲得性免疫應(yīng)答,其分泌的多種細(xì)胞因子對(duì)先天免疫和獲得性免疫又具有調(diào)節(jié)作用。巨噬細(xì)胞功能異常與腫瘤和肥胖等多種疾病密切相關(guān),因而深入研究巨噬細(xì)胞發(fā)育和功能的調(diào)控,對(duì)于進(jìn)一步闡明穩(wěn)態(tài)維持和免疫應(yīng)答的發(fā)生機(jī)制,以及多種疾病的臨床治療都具有重要的意義。 Notch信號(hào)通路在進(jìn)化上高度保守,通過局部細(xì)胞間的相互作用以控制細(xì)胞命運(yùn),是調(diào)控胚胎發(fā)育和多種成體組織器官體內(nèi)穩(wěn)態(tài)和細(xì)胞分化的重要信號(hào)通路。哺乳動(dòng)物表達(dá)四個(gè)Notch受體(Notch1-4)和五個(gè)配體(Jagged1、Jagged2、和Delta-like-1、3、4)。細(xì)胞表面的Notch受體與配體結(jié)合后,在蛋白水解作用下釋放胞內(nèi)段(NICD)轉(zhuǎn)位進(jìn)入細(xì)胞核,與轉(zhuǎn)錄因子RBP-J相互作用,轉(zhuǎn)而募集轉(zhuǎn)錄共激活物,活化下游靶基因的轉(zhuǎn)錄表達(dá)。 Notch信號(hào)通路是調(diào)控髓系造血細(xì)胞發(fā)育的重要途徑,在造血干細(xì)胞的自我更新和髓系細(xì)胞的定向分化中都具有重要作用。同時(shí)Notch信號(hào)通路參與多種免疫細(xì)胞功能的調(diào)控,包括T細(xì)胞、B細(xì)胞和DC等。目前的研究提示,Notch信號(hào)通路對(duì)單核巨噬細(xì)胞的發(fā)育和功能具有重要的意義,但研究較少,而且結(jié)論不明確。因此,本課題同時(shí)應(yīng)用條件性RBP-J基因剔除小鼠模型和巨噬細(xì)胞系RAW264.7穩(wěn)定轉(zhuǎn)染細(xì)胞模型,主要分析了Notch信號(hào)通路對(duì)巨噬細(xì)胞的增殖、抗原呈遞功能和巨噬細(xì)胞活化的影響。主要研究結(jié)果如下: 1.獲得了RBP-J條件性基因剔除小鼠,建立了小鼠巨噬細(xì)胞系RAW264.7的穩(wěn)定轉(zhuǎn)染Notch-1NICD和RBP-J(R218H)的細(xì)胞株。 通過對(duì)RBP-Jflox/wr和Mx-Cre轉(zhuǎn)基因小鼠的繁育,獲得了足夠數(shù)量的的Mx-Cre-RBP-Jflox/wr和Mx-Cre-RBP-Jflox/flox轉(zhuǎn)基因小鼠,給予腹腔注射12次poly I:C誘導(dǎo)Cre重組酶的表達(dá)后,成功獲得了RBP-J基因剔除小鼠。應(yīng)用脂質(zhì)體法轉(zhuǎn)染小鼠巨噬細(xì)胞系RAW264.7,經(jīng)G418篩選和單克隆化,建立了穩(wěn)定轉(zhuǎn)染細(xì)胞株RAW-NIC和RAW-R218H,經(jīng)q-PCR檢測(cè)Notch通路下游基因HES-1表達(dá)水平證實(shí),過表達(dá)Notch-1胞內(nèi)段的RAW-NIC細(xì)胞可以有效激活Notch信號(hào)通路,而過表達(dá)RBP-J顯性負(fù)突變蛋白R(shí)BP-J(R218H)的RAW-R218H細(xì)胞Notch信號(hào)通路抑制,成功獲得了Notch信號(hào)通路活化和阻斷的巨噬細(xì)胞系。RBP-J基因剔除小鼠和穩(wěn)定轉(zhuǎn)染細(xì)胞系的獲得為后續(xù)研究建立了動(dòng)物模型和細(xì)胞模型。 2. Notch信號(hào)通路阻斷后分化成熟的小鼠腹腔巨噬細(xì)胞總數(shù)和比例無明顯異常,Notch信號(hào)通路體外不影響巨噬細(xì)胞系的生長(zhǎng)和增殖。 分離培養(yǎng)RBP-J剔除小鼠腹腔巨噬細(xì)胞,流式細(xì)胞術(shù)檢測(cè)CD11b和F4/80雙陽性細(xì)胞總數(shù)和比例,與對(duì)照小鼠相比,RBP-J剔除后小鼠腹腔巨噬細(xì)胞總數(shù)和比例都無明顯差別;應(yīng)用GSI體外阻斷WT小鼠腹腔巨噬細(xì)胞的Notch通路后,腹腔巨噬細(xì)胞總數(shù)和比例也無明顯異常。應(yīng)用MTT法測(cè)定巨噬細(xì)胞穩(wěn)定轉(zhuǎn)染株的生長(zhǎng)曲線,同時(shí)應(yīng)用流式細(xì)胞術(shù)細(xì)胞分析周期,各組細(xì)胞均無明顯差異。這些結(jié)果表明,Notch信號(hào)阻斷并不影響小鼠腹腔巨噬細(xì)胞分化產(chǎn)生的總數(shù)和比例,Notch信號(hào)通路的體外活化或抑制并不影響巨噬細(xì)胞的生長(zhǎng)和增殖。 3. Notch信號(hào)通路調(diào)控小鼠巨噬細(xì)胞的抗原呈遞功能,這一作用可能是通過調(diào)控共刺激分子CD80和CD86的表達(dá)介導(dǎo)的。 小鼠腹腔巨噬細(xì)胞在RBP-J基因剔除或應(yīng)用GSI體外阻斷Notch信號(hào)通路后,體外與CFSE標(biāo)記的初始T淋巴細(xì)胞共培養(yǎng),流式細(xì)胞術(shù)分析表明增殖的T細(xì)胞總數(shù)和比例顯著低于對(duì)照組。進(jìn)一步研究發(fā)現(xiàn)Notch信號(hào)阻斷后腹腔巨噬細(xì)胞膜表面共刺激分子CD80和CD86表達(dá)顯著減低。而且,巨噬細(xì)胞系Notch信號(hào)通路活化后,可以促進(jìn)更多的T細(xì)胞增殖。這些結(jié)果表明,Notch信號(hào)通路可能通過調(diào)控小鼠巨噬細(xì)胞膜表面共刺激分子CD80和CD86的表達(dá),調(diào)控巨噬細(xì)胞的抗原呈遞功能。 4. Notch信號(hào)通路阻斷后,LPS不能誘導(dǎo)小鼠巨噬細(xì)胞的經(jīng)典活化,Notch信號(hào)參與調(diào)控巨噬細(xì)胞對(duì)LPS應(yīng)答中多種炎性細(xì)胞因子的產(chǎn)生。 Notch信號(hào)通路參與LPS誘導(dǎo)的巨噬細(xì)胞活化,q-PCR證實(shí)LPS活化后,巨噬細(xì)胞多個(gè)Notch受體和配體的表達(dá)改變。RBP-J基因剔除或應(yīng)用GSI體外阻斷Notch信號(hào)通路后,小鼠腹腔巨噬細(xì)胞對(duì)LPS的應(yīng)答改變,與對(duì)照相比,IL-12產(chǎn)生顯著減少,而IL-10產(chǎn)生輕度增加,不能活化為M1型巨噬細(xì)胞,而接近M2巨噬細(xì)胞的表型。此外,Notch信號(hào)通路可以促進(jìn)LPS誘導(dǎo)的巨噬細(xì)胞產(chǎn)生TNF-α和iNOS,抑制IL-1β的產(chǎn)生,阻斷Notch信號(hào)后IL-6的產(chǎn)生增多。這些結(jié)果表明,LPS誘導(dǎo)巨噬細(xì)胞經(jīng)典活化過程中依賴Notch通路的作用,Notch信號(hào)的缺失導(dǎo)致巨噬細(xì)胞M1型活化障礙,而Notch信號(hào)通路與LPS共同調(diào)控巨噬細(xì)胞多種炎性因子的產(chǎn)生。 5. Notch信號(hào)通路調(diào)控巨噬細(xì)胞CD11b的表達(dá)。 通過剔除RBP-J或體外應(yīng)用GSI阻斷小鼠腹腔巨噬細(xì)胞的Notch信號(hào)通路,巨噬細(xì)胞表達(dá)的CD11b在蛋白水平和mRNA水平均顯著減低;活化巨噬細(xì)胞系的Notch信號(hào)通路可以上調(diào)CD11bmRNA水平和蛋白水平的表達(dá)。進(jìn)一步將小鼠CD11b啟動(dòng)子區(qū)克隆構(gòu)建基因報(bào)告質(zhì)粒,通過報(bào)告基因?qū)嶒?yàn)證實(shí),Notch信號(hào)通路對(duì)CD11b基因的轉(zhuǎn)錄無直接調(diào)控作用,可能通過間接作用調(diào)控CD11b的表達(dá)。 結(jié)論 通過本課題的研究證實(shí),Notch信號(hào)通路可以調(diào)控巨噬細(xì)胞的多種生物學(xué)特征。巨噬細(xì)胞發(fā)育過程中,Notch信號(hào)通路的阻斷不影響小鼠腹腔巨噬細(xì)胞分化產(chǎn)生的細(xì)胞總數(shù)和比例,體外實(shí)驗(yàn)中Notch信號(hào)通路不調(diào)控巨噬細(xì)胞的生長(zhǎng)和增殖。然而,Notch信號(hào)通路可以調(diào)控小鼠巨噬細(xì)胞的抗原呈遞功能,這一作用可能是通過調(diào)控共刺激分子CD80和CD86的表達(dá)介導(dǎo)的。LPS誘導(dǎo)巨噬細(xì)胞經(jīng)典活化過程中依賴Notch通路的作用,Notch信號(hào)的缺失導(dǎo)致巨噬細(xì)胞M1型活化障礙,而且Notch信號(hào)通路與LPS共同調(diào)控巨噬細(xì)胞多種炎性因子的產(chǎn)生。此外,Notch信號(hào)通路還可能通過間接作用調(diào)控巨噬細(xì)胞膜表面重要標(biāo)記分子CD11b的表達(dá),這一作用可能成為Notch信號(hào)途徑參與調(diào)控巨噬細(xì)胞發(fā)育和功能的另一靶點(diǎn)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【共引文獻(xiàn)】

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相關(guān)博士學(xué)位論文 前6條

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相關(guān)碩士學(xué)位論文 前10條

1 曾鳳輝;家蠶新基因BmEm4的克隆，表達(dá)與功能研究[D];浙江理工大學(xué);2010年

2 李建杰;骨髓間充質(zhì)干細(xì)胞向神經(jīng)元方向誘導(dǎo)分化及其機(jī)制的實(shí)驗(yàn)研究[D];華北煤炭醫(yī)學(xué)院;2010年

3 劉洪翠;Notch信號(hào)通路對(duì)小鼠小膠質(zhì)細(xì)胞功能的調(diào)控作用[D];第四軍醫(yī)大學(xué);2011年

4 張競(jìng)文;通過Tet-on系統(tǒng)研究Hes1基因在調(diào)控肝干細(xì)胞膽向分化中的作用[D];寧夏醫(yī)科大學(xué);2011年

5 田棣;Hes1基因在小鼠肝干細(xì)胞分化中的功能研究[D];第二軍醫(yī)大學(xué);2006年

6 李鐵民;中藥復(fù)方抑瘤飲體內(nèi)影響小鼠S180腫瘤組織中巨噬細(xì)胞浸潤及TNF-α、iNOS、Bcl-2、Bax表達(dá)的動(dòng)態(tài)研究[D];河北醫(yī)科大學(xué);2006年

7 陶軼;鼠胚神經(jīng)干細(xì)胞的體外培養(yǎng)及其定向分化的實(shí)驗(yàn)研究[D];南京醫(yī)科大學(xué);2006年

8 王劍博;神經(jīng)干細(xì)胞在其分化過程中bHLH基因的表達(dá)變化[D];第四軍醫(yī)大學(xué);2007年

9 尹鄲丹;Notch1對(duì)慢性粒細(xì)胞白血病細(xì)胞系K562的影響[D];第四軍醫(yī)大學(xué);2007年

10 王帥;流式細(xì)胞儀檢測(cè)大鼠下肢深靜脈血栓形成不同時(shí)間FXIIIA的表達(dá)水平[D];大連醫(yī)科大學(xué);2008年

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