【作者】 黎丽；

【导师】 肖建华； 曾辉；

【作者基本信息】 南华大学 ， 免疫学， 2010， 硕士

【摘要】 目的:由于缺乏有效的分离方法,目前对于髓系来源的免疫抑制细胞（Myeloid derived suppressor cells, MDSCs）的研究均停留在混合细胞水平。本实验旨在利用Gfi1:GFP基因敲入小鼠感染性休克模型,采用流式细胞分选技术分离骨髓单核系和粒系来源的MDSCs,并对其表型和体外免疫抑制功能加以鉴定。方法:1)感染性休克小鼠模型的构建:利用LPS连续腹腔注射方法构建Gfi1:GFP基因敲入小鼠感染性休克模型,分别于注射后第4天、第7天检测模型小鼠血浆细胞因子分泌情况,以鉴定造模是否成功;2)骨髓单核系、粒系来源的MDSCs的分离:采用流式细胞分选技术,分离感染性休克小鼠模型骨髓中单核来源的MDSCs (CD11b⁺Gr1^med GFP^-)和粒系来源的MDSCs (CD11b⁺Gr1^med GFP⁺)两群细胞,并进行形态学鉴定;3)表型和功能鉴定:动态观察单核来源的MDSCs (CD11b⁺Gr1^med GFP^-)和粒系来源的MDSCs (CD11b⁺Gr1^med GFP⁺)两群细胞在模型小鼠骨髓细胞中的比例变化和表型差异,并利用体外共培养技术,将分离纯化的单核系和粒系来源的MDSCs亚群分别与CFSE标记的活化CD4+T细胞共培养,体外检测二者对CD4+T细胞增殖的抑制功能,同时采用real-time PCR方法检测二者促炎因子IFN-γ和抑炎因子IL-4、IL-10、IL-13、TGF-β等细胞因子的表达情况,以及发挥免疫抑制作用所需的精氨酸酶（ArginaseⅠ,ArgⅠ）和一氧化氮合酶2（nitric oxide synthetase2, NOS2）的表达水平。结果:1)与正常对照小鼠相比,感染急性期（LPS腹腔注射24小时）模型小鼠外周血各种促炎、抑炎以及趋化因子均明显增高;与急性感染模型小鼠相比,感染性休克小鼠模型（LPS连续腹腔注射7天）外周血血浆中各种促炎因子TNF-α、IL-6、IFN-γ、趋化因子MCP-1水平明显降低,而抑炎因子IL-10的水平略有下降,但仍明显高于正常对照小鼠;2)利用Gfi1:GFP转基因小鼠,根据Gfi1基因在单核系、粒系来源的MDSCs中的差异表达,可分选出纯度大于99%的骨髓单核系来源的MDSCs(其表型为CD11b⁺Gr1^med GFP^-)和粒系来源的MDSCs(其表型为CD11b⁺Gr1^med GFP⁺),经Wright-Giemsa染色形态学鉴定, CD11b⁺Gr1^med GFP+细胞主要为以中晚幼粒细胞为主的幼稚粒细胞;而CD11b⁺Gr1^med GFP-细胞主要是幼稚单核细胞;3)感染性休克模型小鼠骨髓中CD11b⁺的髓系细胞较正常小鼠明显增多,在这些增多的髓系细胞中,尤以粒系来源的MDSCs为主。与正常小鼠相比, LPS连续腹腔注射4天模型小鼠骨髓粒系来源的MDSCs细胞膜表面CD124（IL-4受体）、CD210（IL-10受体）、以及Toll样受体TLR-2和TLR-4的表达有所增加,CD62L表达有所下降,而CD80和CD86的表达则无明显差别;单核系来源的MDSCs细胞表面TLR-2表达减弱, CD124、CD210以及TLR-4表达增强,其他分子表达无显著差异; LPS连续腹腔注射7天模型小鼠骨髓粒系来源的MDSCs细胞膜表面CD124、CD210和TLR-2的表达有所增加,而CD80和CD86的表达无显著差异;单核系来源的MDSCs细胞表面TLR-2表达减弱,其他表型无明显差别;CD11b⁺Gr1^med GFP⁺与CD11b⁺Gr1^med GFPˉ细胞均能不同程度地影响经CD3/28刺激活化的CD4+T淋巴细胞增殖,且该抑制作用随共培养体系中MDSCs比例的增加而呈现递增现象。粒系和单核系来源的骨髓MDSCs细胞均能降低CD4⁺T细胞的增殖活性,且尤以粒系来源的MDSCs的增殖抑制作用显著;粒系来源的MDSCs(CD11b⁺Gr1^med GFP⁺)和单核系来源的MDSCs(CD11b⁺Gr1^med GFP-)细胞亚群的IL-4、IL-10、IL-13、IFN-γ等细胞因子以及ArgⅠ、NOS2等酶的RNA水平表达均增强,说明两个细胞亚群均可通过分泌各种抑制性细胞因子以及这两种酶参与感染性休克免疫抑制。结论:1.利用LPS腹腔连续给药的方法,成功建立了感染性休克小鼠模型;2.利用Gfi1:GFP基因敲入小鼠,采用流式细胞分选技术,可以获得高纯度单核系和粒系来源的骨髓MDSCs;3.感染性休克小鼠模型中,骨髓MDSCs显著增高,且以粒系来源的MDSCs为主;4.感染性休克模型小鼠骨髓粒系来源的MDSCs(CD11b⁺Gr1^med GFP⁺)和单核系来源的MDSCs(CD11b⁺Gr1^med GFP-)两个细胞亚群均能抑制CD4⁺T细胞增殖;且两群细胞均能上调抑炎因子IL-4、IL-10、IL-13和发挥免疫抑制作用的关键酶ArgI、NOS2的表达。更多还原

【Abstract】 Objectives:For lacking of the effective separation methods, most of the studys on MDSCs still keep in a mixed cellular levels. Our experiments were designed to isolate monocyte and granulocyte derived MDSCs （Myeloid-derived suppressor cells, MDSCs） from bone marrow of septic shock Gfi1: GFP knock-in mice, and to further identify their phenotypes and immune suppressive functions in vitro.Methods:1) We seted up Gfi1: GFP knock-in mice septic shock models through continually intraperitoneal injection of LPS （10mg/kg） and detect the concentration of plasma cytokines. 2) We isolate the monocyte(CD11b⁺Gr1^med GFP-)and granulocyte (CD11b⁺Gr1^med GFP⁺) derived MDSCs from septic shock mice bone marrow by the technology of flow cytometry sorting system, and identified their morphology with Wright-Giemsa staining. 3) We observed the proportions and phenotypes of monocyte and granulocyte derived MDSCs in septic shock mice with FACS and identify their immunosuppressive functions with co-culturing of CFSE-labeled CD4+ T cells in different ratios. The expression of the pro-inflammatory, anti-inflammatory factors, ArginaseⅠ（ArgⅠ）and nitric oxide synthetase 2 （ NOS2） within the above MDSCs subsets were also determined with Realtime PCR.Results:1) Comparing with the normal mice, the concentration of the pro-inflammatory and anti-inflammation cytokines increased within the plasma of acute-phase infection （LPS intraperitoneal injection for 24 hours）; the pro-inflammatory factors decreased significantly until LPS intraperitoneal injection for 7 days. Accordingly, the level of anti-inflammatory factor IL-10 reduced slightly but still much higher than that in normal controls. 2) Monocyte and granulocyte derived MDSCs can be separated with BD FACS Aria accoding to the different expression of transcriptional factor Gfi1. We have successfully isolated these two MDSC subsets from septic shock mice, with the phenotype of CD11b⁺Gr1^med GFP^- for monocyte derived MDSC and CD11b⁺Gr1^med GFP+ for granulocyte derived MDSCs respectively. The morphology analysis identified their lineage features. 3) The CD11b⁺Gr1^med myeloid cells in septic shock bone marrows were increased obviously. The most important of all, the main part of the increased cells was granulocyte derived MDSCs. 4) Comparing with the normal control, the expression of CD124, CD210, TLR-2 and TLR-4 were up-regulated and CD62L was down-regulated in granulocyte derived MDSCs from septic shock models , no obvious changes of CD80 and CD86 were observed; the expression patterns in monocyte derived MDSCs were similar to the above, except for the slight down-regulation of TLR2. 5) The CD4+T cells proliferation suppression experiments indicated that both granulocyte and monocyte derived MDSCs possessed immunosuppressive functions in vivo. 6)The expression of anti-inflammatory factors IL-4、IL-10、IL-13 and key enzymes ArgⅠand NOS2 were elevated significantly in both MDSCs subsets. However, TGF-β1 expression was down-regulated slightly.Conclusions:1. We had successfully constructed a septic shock model with Gfi1: GFP knock-in mice （Gfi1GFP / +） and LPS intraperitoneal injection successively.2. We have obtained highly purified monocyte and granulocyte derived MDSCs from bone marrow of septic shock Gfi1GFP / + mice3. The proportions of MDSCs in septic shock mice elevated significantly, and most of which were granulocyte derived MDSCs.4. Both monocyte and granulocyte derived MDSCs could suppress the proliferation of activated CD4+T cells and up-regulate the expression of anti-inflammatory factor IL-4, IL-10, IL-13 and enzymes ArgI、NOS2.更多还原