發(fā)布時(shí)間：2018-01-21 03:07

  本文關(guān)鍵詞： Jagged-1 樹突狀細(xì)胞 T細(xì)胞 細(xì)胞因子 NICD Hes-1 Deltex-1 小鼠　出處：《暨南大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:探討可溶性Jagged-1/Fc嵌合蛋白(Jagged-1)對重組小鼠粒細(xì)胞-巨噬細(xì)胞集落刺激因子(rmGM-CSF)和白細(xì)胞介素4(rmIL-4)體外誘導(dǎo)的小鼠骨髓來源樹突狀細(xì)胞(DCs)的分化和成熟的影響。方法:建立體外誘導(dǎo)小鼠骨髓來源DCs的模型,顯微鏡下觀察rmGM-CSF和rmIL-4協(xié)同誘導(dǎo)的小鼠骨髓DCs形態(tài)學(xué)變化的影響;熒光標(biāo)記單克隆抗體染色技術(shù)結(jié)合流式細(xì)胞儀檢測Jagged-1對小鼠骨髓DCs分化標(biāo)記CD11c及DCs成熟標(biāo)記MHC-Ⅱ、CD86、CD80和CD40表達(dá)的影響;通過westernblot檢測Jagged-1、γ分泌酶抑制劑DAPT(Theγ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alany1]-S-phenylg lycinetbutyl ester,DAPT)、細(xì)菌脂多糖(lipopolysaccharide,LPS)、酵母聚糖A(Zymosan A)對DCs的Jagged-1-Notch下游通路NICD、Hes-1、Deltex-1表達(dá)的影響;通過Luminex蛋白液相芯片技術(shù)和ELISA檢測經(jīng)上述不同處理的DCs和淋巴結(jié)細(xì)胞培養(yǎng)體系上清中白細(xì)胞介素4(IL-4)、白細(xì)胞介素6(IL-6)、白細(xì)胞介素10(IL-10)、白細(xì)胞介素2(IL-2)、白細(xì)胞介素12(IL-12)、干擾素γ(IFN-γ)、轉(zhuǎn)化生長因子β(TGF-β)、腫瘤壞死因子α(TNF-α)的表達(dá)水平。結(jié)果:rmGM-CSF和rmIL-4處理2天骨髓來源細(xì)胞形成明顯大小不等的半貼壁集落,5天后集落開始變小,細(xì)胞表面出現(xiàn)小突起,細(xì)胞逐漸增大,但是培養(yǎng)后期不同處理組細(xì)胞形態(tài)稍有不同。Jagged-1誘導(dǎo)的細(xì)胞具有典型成熟DCs的形態(tài)特征,培養(yǎng)第7天時(shí),Jagged-1組CD11c高表達(dá)細(xì)胞明顯增多,與LPS組無顯著差異,CD86、CD80、CD40和MHC-Ⅱ高表達(dá)細(xì)胞的陽性率也均高于對照組,與LPS、Zymosan A組相似;DCs在加入Jagged-1后1h NICD(Notch Intracellular Domain)開始表達(dá),6h達(dá)高峰,持續(xù)到24h,之后表達(dá)有所下降,而Hes-1、Deltex-1在加入Jagged-1后3h才開始有較高表達(dá),24h達(dá)高峰,持續(xù)到48h,DAPT對Jagged-1-Notch通路有顯著的抑制作用,LPS、Zymosan A組NICD、Hes-1、Deltex-1蛋白表達(dá)不同時(shí)段均較低。骨髓來源細(xì)胞在培養(yǎng)第6天用不同藥物處理后,LPS和Zymosan A能普遍上調(diào)各種細(xì)胞因子的表達(dá),但對TGF-β影響較小,而Jagged-1能大量上調(diào)IL-4、IL-10的表達(dá),并抑制TNF-α表達(dá),對其他細(xì)胞因子影響較小,DAPT對Jagged-1誘導(dǎo)的IL-12和TNF-α表達(dá)抑制有逆轉(zhuǎn)作用,但對其他細(xì)胞因子表達(dá)影響較小。淋巴細(xì)胞經(jīng)過不同濃度的Jagged-1的處理4天后,各種細(xì)胞因子表現(xiàn)出不同的表達(dá)趨勢。與對照組比較,Jagged-1組除TGF-β、IL-6外各種細(xì)胞因子表達(dá)普遍下調(diào),TNF-α、IFN-γ、IL-4水平顯著降低,LPS、Zymosan A組與Jagged-1組相比TNF-α、IFN-γ、IL-6、IL-10、IL-12顯著增加,且LPS組TGF-β顯著減少�；旌狭馨图�(xì)胞反應(yīng)結(jié)果顯示Jagged-1減弱了DCs對同種異基因淋巴細(xì)胞的激活能力。結(jié)論:Jagged-1能通過NICD激活Notch通路下游因子Hes-1、Deltex-1的表達(dá),誘導(dǎo)骨髓來源DCs的分化和成熟,促進(jìn)IL-4、IL-10等Th2型細(xì)胞因子表達(dá)并抑制IFN-γ、IL-12和TNF-α等Th1型細(xì)胞因子表達(dá)。
[Abstract]:Objective: To investigate the effects of soluble Jagged-1/Fc chimeric protein (Jagged-1) colony-stimulating factor on recombinant mouse granulocyte macrophage set (rmGM-CSF) and interleukin 4 (rmIL-4) of murine bone marrow derived dendritic cells in vitro induced (DCs) influence the differentiation and maturation. Methods: to establish the induced mouse bone marrow-derived DCs in vitro model, influence changes of mouse bone marrow DCs microscope observation of rmGM-CSF and rmIL-4 co induced; fluorescent labeled monoclonal antibody staining technique combined with flow cytometry Jagged-1 on mouse bone marrow DCs differentiation marker CD11c and DCs marker of mature MHC- II, CD86, expression of CD80 and CD40; Jagged-1 was detected by Westernblot, gamma secretase inhibitor DAPT (The inhibitor N-[N- y -secretase (3,5-difluorophenacetyl) -1-alany1]-S-phenylg lycinetbutyl ester, DAPT), lipopolysaccharide (lipopolysaccharide, LPS), poly yeast A (Zymosan A) Jagged-1-Notch to DCs downstream NICD, Hes-1, Deltex-1 expression; Luminex protein by liquid chip technology and ELISA detection by the different treatment of DCs and lymph node cell culture system in supernatants of interleukin 4 (IL-4), interleukin 6 (IL-6), white interleukin 10 (IL-10), interleukin 2 (IL-2), interleukin 12 (IL-12) and interferon gamma (IFN- y), transforming growth factor beta (TGF- beta), tumor necrosis factor alpha (TNF- alpha). Results: the expression level of rmGM-CSF and rmIL-4 on the 2 day of bone marrow the size of the cells formed obvious semi adherent colony, 5 days after the colony became small, small protuberances appeared on cell surface, cells increased gradually, but the late culture of different treatment groups have slightly different cell morphology induced by.Jagged-1 cells with typical morphological features of mature DCs, cultured for seventh days, Jagged-1 group of CD11c high expression cell Increased significantly, no significant difference with LPS group, CD86, CD80, high expression of CD40 and MHC- II positive cell rates were also higher than that of the control group, A group and LPS Zymosan, similar to DCs; after joining the Jagged-1 1H NICD (Notch Intracellular Domain) expression peaked at 6h, until 24h, then the expression is while Hes-1, Deltex-1 decreased after joining Jagged-1 3H began to have high expression, peaked at 24h, until 48h, DAPT inhibited significantly on Jagged-1-Notch pathway LPS, Zymosan group A NICD, Hes-1, Deltex-1 protein expression in different periods were lower. Bone marrow cells with different drug treatments in culture for sixth days after LPS, Zymosan and A can express the general increase of various kinds of cytokines, but had little effect on TGF- and Jagged-1 beta, a large number of up-regulated IL-4, IL-10 expression and inhibit TNF- expression, but has little influence on other cytokines, DAPT induced by Jagged-1 IL-12 and TNF- Expression of reverse inhibition, but had little effect on the expression of other cytokines. Lymphocytes treated by different concentrations of Jagged-1 for 4 days, all kinds of cytokines showed different expression trend. Compared with the control group, in Jagged-1 group, TGF- beta, IL-6 expression of various cytokines generally lower, TNF- alpha, IFN- gamma, IL-4 the level of LPS, Zymosan decreased significantly in A group compared with Jagged-1 group, TNF- alpha, IFN- gamma, IL-6, IL-10, IL-12 increased significantly, and LPS group TGF- beta decreased significantly. The mixed lymphocyte reaction showed that Jagged-1 reduced DCs of allogeneic gene activation ability of lymphocytes. Conclusion: Jagged-1 can activate the Notch pathway downstream factor Hes-1 by NICD, the expression of Deltex-1, induce differentiation and maturation of bone marrow-derived DCs promote IL-4, IL-10 and Th2 type cytokines and inhibit the expression of IFN- gamma, IL-12 and TNF- etc Th1 type cytokine expression.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號：1450275