Jagged-1信号诱导小鼠骨髓来源树突状细胞的分化和成熟

发布时间：2018-01-21 03:07

本文关键词： Jagged-1 树突状细胞 T细胞细胞因子 NICD Hes-1 Deltex-1 小鼠　出处：《暨南大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的:探讨可溶性Jagged-1/Fc嵌合蛋白(Jagged-1)对重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和白细胞介素4(rmIL-4)体外诱导的小鼠骨髓来源树突状细胞(DCs)的分化和成熟的影响。方法:建立体外诱导小鼠骨髓来源DCs的模型,显微镜下观察rmGM-CSF和rmIL-4协同诱导的小鼠骨髓DCs形态学变化的影响;荧光标记单克隆抗体染色技术结合流式细胞仪检测Jagged-1对小鼠骨髓DCs分化标记CD11c及DCs成熟标记MHC-Ⅱ、CD86、CD80和CD40表达的影响;通过westernblot检测Jagged-1、γ分泌酶抑制剂DAPT(Theγ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alany1]-S-phenylg lycinetbutyl ester,DAPT)、细菌脂多糖(lipopolysaccharide,LPS)、酵母聚糖A(Zymosan A)对DCs的Jagged-1-Notch下游通路NICD、Hes-1、Deltex-1表达的影响;通过Luminex蛋白液相芯片技术和ELISA检测经上述不同处理的DCs和淋巴结细胞培养体系上清中白细胞介素4(IL-4)、白细胞介素6(IL-6)、白细胞介素10(IL-10)、白细胞介素2(IL-2)、白细胞介素12(IL-12)、干扰素γ(IFN-γ)、转化生长因子β(TGF-β)、肿瘤坏死因子α(TNF-α)的表达水平。结果:rmGM-CSF和rmIL-4处理2天骨髓来源细胞形成明显大小不等的半贴壁集落,5天后集落开始变小,细胞表面出现小突起,细胞逐渐增大,但是培养后期不同处理组细胞形态稍有不同。Jagged-1诱导的细胞具有典型成熟DCs的形态特征,培养第7天时,Jagged-1组CD11c高表达细胞明显增多,与LPS组无显著差异,CD86、CD80、CD40和MHC-Ⅱ高表达细胞的阳性率也均高于对照组,与LPS、Zymosan A组相似;DCs在加入Jagged-1后1h NICD(Notch Intracellular Domain)开始表达,6h达高峰,持续到24h,之后表达有所下降,而Hes-1、Deltex-1在加入Jagged-1后3h才开始有较高表达,24h达高峰,持续到48h,DAPT对Jagged-1-Notch通路有显著的抑制作用,LPS、Zymosan A组NICD、Hes-1、Deltex-1蛋白表达不同时段均较低。骨髓来源细胞在培养第6天用不同药物处理后,LPS和Zymosan A能普遍上调各种细胞因子的表达,但对TGF-β影响较小,而Jagged-1能大量上调IL-4、IL-10的表达,并抑制TNF-α表达,对其他细胞因子影响较小,DAPT对Jagged-1诱导的IL-12和TNF-α表达抑制有逆转作用,但对其他细胞因子表达影响较小。淋巴细胞经过不同浓度的Jagged-1的处理4天后,各种细胞因子表现出不同的表达趋势。与对照组比较,Jagged-1组除TGF-β、IL-6外各种细胞因子表达普遍下调,TNF-α、IFN-γ、IL-4水平显著降低,LPS、Zymosan A组与Jagged-1组相比TNF-α、IFN-γ、IL-6、IL-10、IL-12显著增加,且LPS组TGF-β显著减少。混合淋巴细胞反应结果显示Jagged-1减弱了DCs对同种异基因淋巴细胞的激活能力。结论:Jagged-1能通过NICD激活Notch通路下游因子Hes-1、Deltex-1的表达,诱导骨髓来源DCs的分化和成熟,促进IL-4、IL-10等Th2型细胞因子表达并抑制IFN-γ、IL-12和TNF-α等Th1型细胞因子表达。
[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.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【共引文献】