骨髓單核系、粒系來源的髓系抑制細(xì)胞的分離及鑒定
發(fā)布時(shí)間：2019-06-18 16:29
【摘要】： 目的:由于缺乏有效的分離方法,目前對于髓系來源的免疫抑制細(xì)胞(Myeloid derived suppressor cells, MDSCs)的研究均停留在混合細(xì)胞水平。本實(shí)驗(yàn)旨在利用Gfi1:GFP基因敲入小鼠感染性休克模型,采用流式細(xì)胞分選技術(shù)分離骨髓單核系和粒系來源的MDSCs,并對其表型和體外免疫抑制功能加以鑒定。 方法:1)感染性休克小鼠模型的構(gòu)建:利用LPS連續(xù)腹腔注射方法構(gòu)建Gfi1:GFP基因敲入小鼠感染性休克模型,分別于注射后第4天、第7天檢測模型小鼠血漿細(xì)胞因子分泌情況,以鑒定造模是否成功;2)骨髓單核系、粒系來源的MDSCs的分離:采用流式細(xì)胞分選技術(shù),分離感染性休克小鼠模型骨髓中單核來源的MDSCs (CD11b~+Gr1~(med) GFP~-)和粒系來源的MDSCs (CD11b~+Gr1~(med) GFP~+)兩群細(xì)胞,并進(jìn)行形態(tài)學(xué)鑒定;3)表型和功能鑒定:動態(tài)觀察單核來源的MDSCs (CD11b~+Gr1~(med) GFP~-)和粒系來源的MDSCs (CD11b~+Gr1~(med) GFP~+)兩群細(xì)胞在模型小鼠骨髓細(xì)胞中的比例變化和表型差異,并利用體外共培養(yǎng)技術(shù),將分離純化的單核系和粒系來源的MDSCs亞群分別與CFSE標(biāo)記的活化CD4+T細(xì)胞共培養(yǎng),體外檢測二者對CD4+T細(xì)胞增殖的抑制功能,同時(shí)采用real-time PCR方法檢測二者促炎因子IFN-γ和抑炎因子IL-4、IL-10、IL-13、TGF-β等細(xì)胞因子的表達(dá)情況,以及發(fā)揮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)的表達(dá)水平。 結(jié)果:1)與正常對照小鼠相比,感染急性期(LPS腹腔注射24小時(shí))模型小鼠外周血各種促炎、抑炎以及趨化因子均明顯增高;與急性感染模型小鼠相比,感染性休克小鼠模型(LPS連續(xù)腹腔注射7天)外周血血漿中各種促炎因子TNF-α、IL-6、IFN-γ、趨化因子MCP-1水平明顯降低,而抑炎因子IL-10的水平略有下降,但仍明顯高于正常對照小鼠;2)利用Gfi1:GFP轉(zhuǎn)基因小鼠,根據(jù)Gfi1基因在單核系、粒系來源的MDSCs中的差異表達(dá),可分選出純度大于99%的骨髓單核系來源的MDSCs(其表型為CD11b~+Gr1~(med) GFP~-)和粒系來源的MDSCs(其表型為CD11b~+Gr1~(med) GFP~+),經(jīng)Wright-Giemsa染色形態(tài)學(xué)鑒定, CD11b~+Gr1~(med) GFP+細(xì)胞主要為以中晚幼粒細(xì)胞為主的幼稚粒細(xì)胞;而CD11b~+Gr1~(med) GFP-細(xì)胞主要是幼稚單核細(xì)胞;3)感染性休克模型小鼠骨髓中CD11b~+的髓系細(xì)胞較正常小鼠明顯增多,在這些增多的髓系細(xì)胞中,尤以粒系來源的MDSCs為主。與正常小鼠相比, LPS連續(xù)腹腔注射4天模型小鼠骨髓粒系來源的MDSCs細(xì)胞膜表面CD124(IL-4受體)、CD210(IL-10受體)、以及Toll樣受體TLR-2和TLR-4的表達(dá)有所增加,CD62L表達(dá)有所下降,而CD80和CD86的表達(dá)則無明顯差別;單核系來源的MDSCs細(xì)胞表面TLR-2表達(dá)減弱, CD124、CD210以及TLR-4表達(dá)增強(qiáng),其他分子表達(dá)無顯著差異; LPS連續(xù)腹腔注射7天模型小鼠骨髓粒系來源的MDSCs細(xì)胞膜表面CD124、CD210和TLR-2的表達(dá)有所增加,而CD80和CD86的表達(dá)無顯著差異;單核系來源的MDSCs細(xì)胞表面TLR-2表達(dá)減弱,其他表型無明顯差別;CD11b~+Gr1~(med) GFP~+與CD11b~+Gr1~(med) GFPˉ細(xì)胞均能不同程度地影響經(jīng)CD3/28刺激活化的CD4+T淋巴細(xì)胞增殖,且該抑制作用隨共培養(yǎng)體系中MDSCs比例的增加而呈現(xiàn)遞增現(xiàn)象。粒系和單核系來源的骨髓MDSCs細(xì)胞均能降低CD4~+T細(xì)胞的增殖活性,且尤以粒系來源的MDSCs的增殖抑制作用顯著;粒系來源的MDSCs(CD11b~+Gr1~(med) GFP~+)和單核系來源的MDSCs(CD11b~+Gr1~(med) GFP-)細(xì)胞亞群的IL-4、IL-10、IL-13、IFN-γ等細(xì)胞因子以及ArgⅠ、NOS2等酶的RNA水平表達(dá)均增強(qiáng),說明兩個(gè)細(xì)胞亞群均可通過分泌各種抑制性細(xì)胞因子以及這兩種酶參與感染性休克免疫抑制。 結(jié)論: 1.利用LPS腹腔連續(xù)給藥的方法,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,采用流式細(xì)胞分選技術(shù),可以獲得高純度單核系和粒系來源的骨髓MDSCs; 3.感染性休克小鼠模型中,骨髓MDSCs顯著增高,且以粒系來源的MDSCs為主; 4.感染性休克模型小鼠骨髓粒系來源的MDSCs(CD11b~+Gr1~(med) GFP~+)和單核系來源的MDSCs(CD11b~+Gr1~(med) GFP-)兩個(gè)細(xì)胞亞群均能抑制CD4~+T細(xì)胞增殖;且兩群細(xì)胞均能上調(diào)抑炎因子IL-4、IL-10、IL-13和發(fā)揮免疫抑制作用的關(guān)鍵酶ArgI、NOS2的表達(dá)。
[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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 湯耀卿;李磊;;膿毒癥動物模型制作方略及應(yīng)用[J];中華實(shí)驗(yàn)外科雜志;2006年12期
2 朱雪琦;劉清泉;姚詠明;;膿毒癥動物模型制備方法的研究進(jìn)展[J];中國危重病急救醫(yī)學(xué);2006年02期
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