本文選題：Notch信號(hào)途徑 + RBP-J��； 參考：《第四軍醫(yī)大學(xué)》2009年博士論文

【摘要】： 單核細(xì)胞是機(jī)體先天性免疫和獲得性免疫應(yīng)答的重要組成部分,是單核吞噬系統(tǒng)的主要成員之一。大量研究表明骨髓單核細(xì)胞是樹(shù)突狀細(xì)胞和巨噬細(xì)胞的共同前體細(xì)胞,他們共同構(gòu)成了外來(lái)微生物入侵時(shí)機(jī)體的有效防御系統(tǒng),同時(shí)對(duì)于生理狀態(tài)下清除機(jī)體內(nèi)衰老和凋亡的細(xì)胞,維持機(jī)體的穩(wěn)態(tài)發(fā)揮了不可替代的作用。樹(shù)突狀細(xì)胞是目前已知的功能最強(qiáng)大的抗原呈遞細(xì)胞,是體內(nèi)激發(fā)T細(xì)胞免疫應(yīng)答的首要環(huán)節(jié)。而巨噬細(xì)胞能夠吞噬細(xì)菌、病毒、凋亡的細(xì)胞及抗原抗體復(fù)合物等,并通過(guò)胞內(nèi)溶酶體等將其消化清除,同時(shí)巨噬細(xì)胞還具有較強(qiáng)的抗原處理和遞呈能力,能夠通過(guò)分泌多種細(xì)胞因子發(fā)揮免疫調(diào)節(jié)和介導(dǎo)炎癥反應(yīng)。單核細(xì)胞,樹(shù)突狀細(xì)胞以及巨噬細(xì)胞的異常發(fā)育必然導(dǎo)致機(jī)體免疫系統(tǒng)功能和機(jī)體內(nèi)環(huán)境的紊亂,最終引發(fā)機(jī)體的各種疾病。 細(xì)胞的分化是一個(gè)復(fù)雜的生命過(guò)程,其中涉及多種信號(hào)途徑的協(xié)同和調(diào)控。Notch信號(hào)途徑在進(jìn)化中高度保守,從線蟲(chóng)到人,從胚胎發(fā)育到成年個(gè)體的多種系統(tǒng)中都發(fā)揮著重要作用。Notch受體和配體家族均是單次跨膜蛋白,通過(guò)細(xì)胞間的相互作用,Notch受體胞內(nèi)段發(fā)生水解入核,同時(shí)募集并結(jié)合多種蛋白分子形成轉(zhuǎn)錄共激活物,去除RBP-J的轉(zhuǎn)錄抑制作用,調(diào)控下游靶基因的表達(dá)。Notch信號(hào)途徑對(duì)多細(xì)胞生物的很多不同組織中的細(xì)胞命運(yùn)起著決定性作用,例如神經(jīng)系統(tǒng),血管系統(tǒng),造血系統(tǒng)等等。在免疫系統(tǒng)的發(fā)育過(guò)程中,Notch信號(hào)途徑在多個(gè)環(huán)節(jié)參與了免疫細(xì)胞發(fā)育命運(yùn)的選擇,如T/B細(xì)胞的分化、脾臟中邊緣帶B細(xì)胞和濾泡B細(xì)胞的分化等等。同時(shí),Notch信號(hào)途徑也是Th1和Th2免疫反應(yīng)選擇的調(diào)控因素之一。但Notch信號(hào)途徑在其他免疫類型細(xì)胞諸如DC、巨噬細(xì)胞以及NK細(xì)胞發(fā)育的調(diào)控作用尚不十分清楚。雖然有報(bào)道顯示阻斷Notch信號(hào)途徑可以抑制DC的發(fā)育,然而對(duì)于調(diào)控過(guò)程中的分子機(jī)制卻是知之甚少。 為了更加全面闡明Notch信號(hào)途徑在免疫系統(tǒng)發(fā)育過(guò)程的調(diào)控作用,本課題利用條件性基因剔除技術(shù),在小鼠造血干細(xì)胞中誘導(dǎo)剔除Notch信號(hào)途徑的關(guān)鍵性轉(zhuǎn)錄因子RBP-J,在造血系細(xì)胞中徹底阻斷Notch信號(hào)途徑。通過(guò)RBP-J剔除后對(duì)單核細(xì)胞,DC和巨噬細(xì)胞發(fā)育及功能變化的深入探討,進(jìn)一步明確了Notch信號(hào)途徑調(diào)控單核細(xì)胞命運(yùn)選擇中的作用,以及對(duì)DC和巨噬細(xì)胞功能的影響,并分析了相關(guān)的分子機(jī)制。 主要研究成果如下: 1.通過(guò)交配Mx-Cre轉(zhuǎn)基因小鼠和RBP-Jflox/flox條件基因剔除小鼠,最終得到了同時(shí)攜帶Mx-Cre×RBP-Jflox/flox的雙轉(zhuǎn)基因條件剔除小鼠。經(jīng)poly I-C誘導(dǎo)8-12周后得到RBP-J基因剔除小鼠。經(jīng)Southern blot鑒定,骨髓中RBP-J剔除效率約接近100%。通過(guò)骨髓移植實(shí)驗(yàn)表明,剔除RBP-J使單核細(xì)胞在體內(nèi)向DC方向的分化受到明顯抑制,而向巨噬細(xì)胞方向的分化得到加強(qiáng)。體外的細(xì)胞誘導(dǎo)分化實(shí)驗(yàn)也印證了體內(nèi)的實(shí)驗(yàn)結(jié)果。分子生物學(xué)檢測(cè)表明RBP-J-/-巨噬細(xì)胞表達(dá)M-CSFR水平明顯升高,提示Notch信號(hào)途徑可能是通過(guò)直接或間接調(diào)控M-CSFR的表達(dá)調(diào)控單核細(xì)胞的分化方向。 2.在DC中剔除RBP-J后,用LPS將不能有效刺激DC的發(fā)育成熟,DC發(fā)育停留在突起較少、MHCII表達(dá)較低的不成熟階段,同時(shí)其遷移和抗原呈遞功能受到損傷。進(jìn)一步研究表明,趨化因子受體CXCR4是Notch信號(hào)途徑下游的間接靶分子,在DC的發(fā)育過(guò)程中可以促進(jìn)DC突起的長(zhǎng)出和MHCII分子的表達(dá)。用慢病毒在RBP-J-/-DC中過(guò)表達(dá)CXCR4可以挽救由阻斷Notch信號(hào)途徑所引起的缺陷表型。我們的研究結(jié)果首次揭示了Notch信號(hào)可以通過(guò)調(diào)控CXCR4促進(jìn)DC發(fā)育成熟。 3.剔除RBP-J后,巨噬細(xì)胞在LPS刺激后不能向M1型巨噬細(xì)胞極化,而是被極化為M2型巨噬細(xì)胞,其遷移、吞噬、抗原加工以及呈遞能力減退,失去了維持機(jī)體穩(wěn)態(tài)的功能。在腫瘤中,RBP-J-/-巨噬細(xì)胞腫瘤殺傷功能減弱,腫瘤重量和體積沒(méi)有因?yàn)榫奘杉?xì)胞的存在受到抑制。免疫組化結(jié)果顯示,RBP-J-/-巨噬細(xì)胞可以促進(jìn)腫瘤中新生血管的生成,表明其具有腫瘤相關(guān)性巨噬細(xì)胞(TAM)的特性。同時(shí),激活巨噬細(xì)胞中的Notch信號(hào)途徑可以增強(qiáng)細(xì)胞的抗原呈遞能力,并激發(fā)較強(qiáng)的Th1型免疫應(yīng)答。這些結(jié)果表明Notch信號(hào)途徑是巨噬細(xì)胞M1/M2極化的關(guān)鍵調(diào)控信號(hào)。 綜上所述,我們首次通過(guò)RBP-J條件性基因剔除小鼠,觀察了Notch/RBP-J信號(hào)對(duì)于小鼠單核細(xì)胞來(lái)源DC和巨噬細(xì)胞發(fā)育和功能的調(diào)控作用。我們的結(jié)果表明,Notch信號(hào)參與調(diào)控單核細(xì)胞發(fā)育命運(yùn)的方向;通過(guò)調(diào)控CXCR4的表達(dá)促進(jìn)DC的成熟;同時(shí)對(duì)于巨噬細(xì)胞向M1方向極化是必須的。以上研究闡明了Notch信號(hào)途徑在天然免疫系統(tǒng)發(fā)育中的重要作用,并充分解釋了其作用機(jī)制,因此具有重要的理論和實(shí)際意義。
[Abstract]:Monocyte is an important part of innate immunity and acquired immune response , which is one of the main members of mononuclear phagocytizing system .



Notch signaling pathway plays an important role in the development of immune system . Notch signaling pathway plays a decisive role in the development of immune cells , such as nervous system , vascular system , hematopoietic system and so on . Notch signaling pathway plays a decisive role in the development of immune cells , such as nervous system , vascular system , hematopoietic system and so on .



In order to elucidate the role of Notch signaling pathway in the development of immune system more fully , this topic uses conditional gene knockout technique to induce the critical transcription factor of the Notch signaling pathway in hematopoietic stem cells of mice . The effect of Notch signaling pathway on monocyte , DC and macrophage development and function changes is further clarified . The effects of Notch signaling pathway on monocyte , DC and macrophage development and function changes are further clarified , and the related molecular mechanism is analyzed .



The main results are as follows :



1 . The mice were knocked out of mice by mating of the transgenic mice and the mouse with the conditional gene , which simultaneously carried the double - transgenic conditions of the mice . The results showed that the differentiation of the monocytes in the direction of the body was nearly 100 % after 8 - 12 weeks of induction .



2 . It is shown that CXCR4 is an indirect target molecule downstream of Notch signaling pathway , and its migration and antigen presentation function are damaged . Further research shows that chemokine receptor CXCR4 is an indirect target molecule downstream of Notch signaling pathway . It is shown that chemokine receptor CXCR4 is an indirect target molecule downstream of Notch signaling pathway .



3 . After LPS stimulation , macrophages cannot be polarized to M1 - type macrophages after LPS stimulation , but are polarized into M2 - type macrophages . Their migration , phagocytosis , antigen processing and presentation ability decrease . The tumor weight and volume are not inhibited by the presence of macrophages . Immunohistochemical results show that the pathway of Notch signaling in macrophages can enhance the antigen presentation ability of cells and stimulate stronger Th1 - type immune responses . These results suggest that Notch signaling pathway is a key regulatory signal for the polarization of macrophages M1 / M2 .



In conclusion , we have observed the role of Notch signal in regulating the development and function of monocytes in mice . The results show that Notch signaling plays an important role in regulating the development of monocytes .

【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】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 卓少元;方肇勤;管冬元;吳中華;高必峰;;活血化瘀法調(diào)節(jié)DEN肝癌大鼠相關(guān)基因的生物信息學(xué)分析[J];遼寧中醫(yī)雜志;2013年12期

相關(guān)碩士學(xué)位論文 前1條

1 陳韻如;Notch信號(hào)途徑對(duì)小鼠樹(shù)突狀細(xì)胞抗原提呈功能和Th1/Th2偏轉(zhuǎn)潛能的影響[D];第四軍醫(yī)大學(xué);2010年

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本文編號(hào)：1833572