骨髓单核系、粒系来源的髓系抑制细胞的分离及鉴定
发布时间：2019-06-18 16:29
【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。
[Abstract]:Objective: As a result of the lack of effective separation method, the research of Myeloid-derived support cells (MDSCs) in myeloid-derived immunosuppressive cells (MDSCs) remained at the level of mixed cells. The purpose of this study was to use the Gfi1: GFP gene to knock into the mouse's septic shock model and to separate the MDSCs from the bone marrow mononuclear cell and the particle system by flow cytometry, and to identify its phenotype and in vitro immunosuppression function. Methods:1) Construction of the model of the septic shock mice: The Gfi1: GFP gene was built into the mice's septic shock model by means of the LPS continuous intraperitoneal injection method, and the secretion of plasma cytokines in the model mice was detected on the 4th and 7th day after injection, so as to identify the model. No success;2) The separation of MDSCs from the bone marrow mononuclear cell and the particle system: the two groups of MDSCs (CD11b + Gr1-(med) GFP--) and MDSCs (CD11b + Gr1-(med) GFP-+) from the bone marrow of the septic shock mouse model were isolated by flow cytometry. (3) Phenotypic and functional identification: The ratio and phenotype of MDSCs (CD11b ~ + Gr1-(med) GFP ~ +) and MDSCs (CD11b ~ + Gr1 ~ (med) GFP ~ +) of single-core-derived MDSCs (CD11b ~ + Gr1 ~ (med) GFP ~ +) in the bone marrow cells of the model mice were observed dynamically and co-cultured in vitro. The proliferation of CD4 + T cells was detected by co-culture with CFSE-labeled activated CD4 + T cells, and the inhibition of the proliferation of CD4 + T cells was detected in vitro, and the two pro-inflammatory factors, IFN-1, and anti-inflammatory factors, IL-4, IL-10, and IL-4 were detected by the real-time PCR. Expression of cytokines such as -13, TGF-1, and the expression of arginase I, Arg I, and nitric oxide synthase 2 (NOS2) required for immunosuppression The results were as follows:1) Compared with normal control mice, the peripheral blood of mice infected with acute phase (LPS-peritoneal injection for 24 hours) was significantly higher in the peripheral blood of mice than in the normal control mice. Compared with the model mice, the levels of TNF-1, IL-6, IFN-1 and MCP-1 in the plasma of the peripheral blood of the septic shock mouse model (LPS for 7 days) were significantly reduced, while the level of the pro-inflammatory factor IL-10 decreased slightly, but was still significantly higher than that of the normal control mice;2 ) Using Gfi1: GFP transgenic mice, according to the differential expression of the Gfi1 gene in the MDSCs of the single-core system and the grain-based source, MDSCs with a purity of more than 99% (whose phenotype is CD11b-+ Gr1-(med) GFP--) and MDSCs of the particle-based source (whose phenotype is CD11b-+ Gr1-(med) GFP-+) is obtained by Wright-Giemsa. The results showed that CD11b ~ + Gr1 ~ (med) GFP + cells were mainly immature granulocytes with middle and late myelocytes, while the CD11b ~ + Gr1 ~ (med) GFP-cells were mainly immature monocytes;3) The number of CD11b ~ + in the bone marrow of mice with septic shock was more than that of normal mice. Of or relating to a source of a grain. The expression of CD124 (IL-4 receptor), CD210 (IL-10 receptor) and Toll-like receptor TLR-2 and TLR-4 increased, and the expression of CD62L decreased, while the expression of CD80 and CD86 decreased compared with normal mice. There was no significant difference between the expression of TLR-2 and the expression of CD124, CD210 and TLR-4, and there was no significant difference in the expression of CD124, CD210 and TLR-4. The expression of CD80 and CD86 increased, but the expression of CD80 and CD86 had no significant difference; the expression of TLR-2 on the surface of MDSCs of single-core system was weakened, and the other phenotypes did not differ significantly; CD11b ~ + Gr1-(med) GFP ~ + and CD11b ~ + Gr1-(med) GFP-expressing cells could affect the CD4 + stimulated by CD3/28. T-lymphocyte proliferation, and the inhibitory effect increases with the ratio of MDSCs in the co-culture system The MDSCs (CD11b ~ + Gr1 ~ (med) GFP ~ +) and MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) of the single-core-derived MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) cell subpopulation of the MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) of the single-core-derived MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) cell subpopulation of the MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) of the single-core source were significantly inhibited. The expression of the isoenzymes such as 0, IL-13, IFN-1, and other enzymes such as Arg I and NOS2 were enhanced, suggesting that both cell subpopulations can be involved in the infection by the secretion of various inhibitory cytokines and the two enzymes. shock Immunosuppression. Conclusion:1. The method of continuous administration of LPS in the abdominal cavity and 2. using the Gfi1: GFP gene to knock the mouse into the mouse, and adopting flow cytometry to obtain the mouse model; Bone marrow MDSCs from high-purity mononuclear and particle-based sources;3. Bone marrow M in an infectious shock mouse model The MDSCs (CD11b ~ + Gr1 ~ (med) GFP ~ +) and MDSCs (CD11b ~ + Gr1 ~ (me) from the rat bone marrow of septic shock model D) Both cell subpopulations of GFP-) can inhibit the proliferation of CD4 ~ + T cells, and both group of cells can increase the anti-inflammatory factor IL-4, IL-10 and IL-10.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2501630
[Abstract]:Objective: As a result of the lack of effective separation method, the research of Myeloid-derived support cells (MDSCs) in myeloid-derived immunosuppressive cells (MDSCs) remained at the level of mixed cells. The purpose of this study was to use the Gfi1: GFP gene to knock into the mouse's septic shock model and to separate the MDSCs from the bone marrow mononuclear cell and the particle system by flow cytometry, and to identify its phenotype and in vitro immunosuppression function. Methods:1) Construction of the model of the septic shock mice: The Gfi1: GFP gene was built into the mice's septic shock model by means of the LPS continuous intraperitoneal injection method, and the secretion of plasma cytokines in the model mice was detected on the 4th and 7th day after injection, so as to identify the model. No success;2) The separation of MDSCs from the bone marrow mononuclear cell and the particle system: the two groups of MDSCs (CD11b + Gr1-(med) GFP--) and MDSCs (CD11b + Gr1-(med) GFP-+) from the bone marrow of the septic shock mouse model were isolated by flow cytometry. (3) Phenotypic and functional identification: The ratio and phenotype of MDSCs (CD11b ~ + Gr1-(med) GFP ~ +) and MDSCs (CD11b ~ + Gr1 ~ (med) GFP ~ +) of single-core-derived MDSCs (CD11b ~ + Gr1 ~ (med) GFP ~ +) in the bone marrow cells of the model mice were observed dynamically and co-cultured in vitro. The proliferation of CD4 + T cells was detected by co-culture with CFSE-labeled activated CD4 + T cells, and the inhibition of the proliferation of CD4 + T cells was detected in vitro, and the two pro-inflammatory factors, IFN-1, and anti-inflammatory factors, IL-4, IL-10, and IL-4 were detected by the real-time PCR. Expression of cytokines such as -13, TGF-1, and the expression of arginase I, Arg I, and nitric oxide synthase 2 (NOS2) required for immunosuppression The results were as follows:1) Compared with normal control mice, the peripheral blood of mice infected with acute phase (LPS-peritoneal injection for 24 hours) was significantly higher in the peripheral blood of mice than in the normal control mice. Compared with the model mice, the levels of TNF-1, IL-6, IFN-1 and MCP-1 in the plasma of the peripheral blood of the septic shock mouse model (LPS for 7 days) were significantly reduced, while the level of the pro-inflammatory factor IL-10 decreased slightly, but was still significantly higher than that of the normal control mice;2 ) Using Gfi1: GFP transgenic mice, according to the differential expression of the Gfi1 gene in the MDSCs of the single-core system and the grain-based source, MDSCs with a purity of more than 99% (whose phenotype is CD11b-+ Gr1-(med) GFP--) and MDSCs of the particle-based source (whose phenotype is CD11b-+ Gr1-(med) GFP-+) is obtained by Wright-Giemsa. The results showed that CD11b ~ + Gr1 ~ (med) GFP + cells were mainly immature granulocytes with middle and late myelocytes, while the CD11b ~ + Gr1 ~ (med) GFP-cells were mainly immature monocytes;3) The number of CD11b ~ + in the bone marrow of mice with septic shock was more than that of normal mice. Of or relating to a source of a grain. The expression of CD124 (IL-4 receptor), CD210 (IL-10 receptor) and Toll-like receptor TLR-2 and TLR-4 increased, and the expression of CD62L decreased, while the expression of CD80 and CD86 decreased compared with normal mice. There was no significant difference between the expression of TLR-2 and the expression of CD124, CD210 and TLR-4, and there was no significant difference in the expression of CD124, CD210 and TLR-4. The expression of CD80 and CD86 increased, but the expression of CD80 and CD86 had no significant difference; the expression of TLR-2 on the surface of MDSCs of single-core system was weakened, and the other phenotypes did not differ significantly; CD11b ~ + Gr1-(med) GFP ~ + and CD11b ~ + Gr1-(med) GFP-expressing cells could affect the CD4 + stimulated by CD3/28. T-lymphocyte proliferation, and the inhibitory effect increases with the ratio of MDSCs in the co-culture system The MDSCs (CD11b ~ + Gr1 ~ (med) GFP ~ +) and MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) of the single-core-derived MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) cell subpopulation of the MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) of the single-core-derived MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) cell subpopulation of the MDSCs (CD11b ~ + Gr1 ~ (med) GFP-) of the single-core source were significantly inhibited. The expression of the isoenzymes such as 0, IL-13, IFN-1, and other enzymes such as Arg I and NOS2 were enhanced, suggesting that both cell subpopulations can be involved in the infection by the secretion of various inhibitory cytokines and the two enzymes. shock Immunosuppression. Conclusion:1. The method of continuous administration of LPS in the abdominal cavity and 2. using the Gfi1: GFP gene to knock the mouse into the mouse, and adopting flow cytometry to obtain the mouse model; Bone marrow MDSCs from high-purity mononuclear and particle-based sources;3. Bone marrow M in an infectious shock mouse model The MDSCs (CD11b ~ + Gr1 ~ (med) GFP ~ +) and MDSCs (CD11b ~ + Gr1 ~ (me) from the rat bone marrow of septic shock model D) Both cell subpopulations of GFP-) can inhibit the proliferation of CD4 ~ + T cells, and both group of cells can increase the anti-inflammatory factor IL-4, IL-10 and IL-10.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 汤耀卿;李磊;;脓毒症动物模型制作方略及应用[J];中华实验外科杂志;2006年12期
2 朱雪琦;刘清泉;姚咏明;;脓毒症动物模型制备方法的研究进展[J];中国危重病急救医学;2006年02期
，本文编号：2501630
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2501630.html
最近更新
教材专著
