髓初始髓源抑制细胞亚群表型鉴定及对T细胞免疫抑制功能的研究-免疫学

1主要英文缩略语索引缩写词 英文全称 中文名称Arg nitric oxide synthetase 精氨酸酶APC antigen presenting cell 抗原提呈细胞BSA bovine serum albumin 牛血清白蛋白CBA cytometric bead array 流式微珠分析CDK4 cyclin-dependent kinase 4 细胞周期蛋白依赖激酶4CFSE carboxytluorescei diacetate succinimidyl este羧基荧光素乙酰乙酸琥珀酰亚胺脂CTLs cytotoxic T lymohocytes 细胞毒性 T 细胞DCs dendritic cells 树突状细胞DEPC diethyl-pryocarbonate 焦碳酸二乙酯EDTA ethylene diamietetraacetic acid 乙二胺四乙酸FACS fluorescence activated cell sorting 荧光活化细胞分选系统FBS fetal bovine serum 胎牛血清FITC fluorescein isothiocynate 异硫氰酸荧光素Gfi growth factor independence 非依赖性生长因子GFP green fluorescent protein 绿色荧光蛋白GM-CSF granulocyte-macrophage colony stimulating factor 粒细胞-巨噬细胞集落刺激因子GVHD graft versus host disease 移植物抗宿主反应H2O2 hydrogen peroxide 过氧化氢2ICAM intercellular cell adhesion molecule 细胞间细胞黏附分子IFN- interferon- 干扰素IL-1 interleukin-1 白细胞介素-1IL-2 interleukin-2 白细胞介素-2IL-4 interleukin-4 白细胞介素-4IL-6 interleukin-6 白细胞介素-6IL-10 interleukin-10 白细胞介素-10IL-12p70 interleukin-12p70 白细胞介素-12p70IL-13 interleukin-13 白细胞介素-13L-Arg L-arginine L-精氨酸LPS lipopolysaccharide 脂多糖MCP-1 monocyte chemoattractant protein-1 单核细胞趋化蛋白-1MDSCs myeloid-derived suppressor cells 髓源抑制细胞MHC major histocompatibility complex 主要组织相容性复合体NO nitric oxide 一氧化氮NOS nitric oxide synthetase 一氧化氮合成酶iNOS inducible nitric oxide synthetase 诱导型一氧化氮合成酶NF-B nuclear factor-B 细胞核因子 NF-BONOO peroxynitrite 过氧亚硝酸盐ROS reactive oxygen species 活性氧rpm revolutions per minute 每分钟转数3RPMI-1640 roswell park memorial institute-1640 1640 Medium RPMI-1640 培养基RT-PCR reverse transcriptase polymerase chain reaction逆转录聚合酶链反应SIRS systemic inflammatory response syndrome 全身炎症反应综合征SPF specified-pathogens free 无特定病原体STAT signal transducer and activator of transcription 信号转导和转录激活子TCR T cell receptor T 细胞受体Th helper T cell 辅助性 T 细胞TNF- tumor necrosis factor- 肿瘤坏死因子-Treg regulatory T cell 调节性 T 细胞TLRs Toll-like receptors Toll 样受体TGF- transforming growth factor- 转化生长因子-PBS phosophate buffered saline 磷酸盐缓冲液PerCP peridinin chlorop hyllprotein 多甲藻黄素叶绿素蛋白PE phycoerythrin 藻红蛋白Nave MDSCnave myeloid-derived suppressor cells初始髓系抑制细胞L-NMMA NG-Methyl-L-arginine acetate salt N 甲基左旋精氨酸醋酸盐L-NIL L-N6-(1-1mindethyl)lysine hydrochlorideL-N6-氢氯赖氨酸nor-NOHA NW-Hydroxy-nor-L-arginine Diacetate sateN 羟基去甲左旋精氨酸双乙酸盐4中文摘要目的： MDSCs 是一群异质性的细胞，通常在肿瘤，感染，炎症和移植等病理情况时增多。一般认为，在病理状态下，MDSCs 对免疫系统具有广泛的抑制作用，发挥调节免疫系统的功能；而正常状态下，MDSCs 是否具有免疫抑制作用尚有争论。目前，多数研究针对肿瘤和感染等病理条件下混合 MDSCs 或MDSCs 的亚群进行研究，而对骨髓初始 MDSCs 的研究相对较少。本实验旨在使用 Gfi1:GFP 基因敲入小鼠模型，从正常模型小鼠的骨髓中分离初始 MDSCs （nave MDSCs） ，运用流式细胞仪（BD FACS Aria）分选不同亚群细胞，采用免疫学方法，研究初始 MDSCs 及其亚群在正常情况下可能具有的表型和 T 细胞免疫抑制功能；重点研究骨髓 nave MDSCs 不同亚群对 T 细胞功能的影响及其机制。方法：通过对 Gfi1：GFP 基因敲入 C57BL/6J 小鼠模型骨髓细胞分离纯化，利用免疫磁珠技术分离骨髓初始 MDSCs(CD11b+细胞)，流式细胞仪检测分选纯度；利用离心涂片法和 Wright Giemsa 染色法对初始 MDSCs 进行形态学分析；将骨髓初始 MDSCs 分别与 CD3/28 Dynabeads T-Activator 预刺激的 CD4+T 或CD8+T 细胞共培养，流式细胞术检测它们对 CD4+T 和 CD8+T 细胞增殖功能的影响。利用流式细胞技术根据 CD11b 、Gr1 和 Gfi1:GFP 表达的差异将 MDSCs 分为不同亚群，并鉴定其表型CD62L、Ly6C、CD204、CD206、CD80、CD86 、TLR4、TLR2、CD124 、CD210 等的表达差异；用离心涂片法和 Wright Giemsa 染色法对不同亚群进行形态学分析；用纯化的骨髓初始 MDSCs 的不同亚群细胞分别与 CD3/28 Dynabeads T-Activator 预刺激的 CD4+T 或 CD8+T 细胞共培养，流式细胞术检测它们对CD4+T 和 CD8+T 细胞增殖功能的影响；并应用 Realtime-PCR 方法检测不同亚群细胞抗炎因子及相关酶 mRNA 的表达，从而初步探讨它们发挥免疫调节作用的分子机制；进而使用相关酶的抑制剂阻断并且应用流式细胞术分析进一步的观察哪一种酶起主要作用。5结果： 骨髓初始 MDSCs 是健康小鼠免疫细胞的重要组成成分，其形态为大小不一、胞质呈淡蓝色、核为分叶形、环形或椭圆形的细胞群；初始 MDSCs 在体外可以抑制 CD4+T 和 CD8+T 细胞增殖，在体内可以延缓和降低小鼠急性肝衰竭模型的死亡率。按 CD11b 、Gr1 和 Gfi1:GFP 等分子表达的差异及细胞形态学差异可将初始 MDSCs 分为四个亚群：CD11b +Gr1+ GFP-（M1） 、CD11blowGr1low GFP+（G1） 、CD11b lowGr1high GFP+（G2 ）及 CD11bhighGr1high GFP+（G3） 。在正常小鼠骨髓细胞中，G1 群细胞占骨髓总细胞的 1.190.38%，占骨髓初始 MDSCs 的 4.040.9%；G2 群细胞占骨髓总细胞的 8.492.69%，占骨髓 MDSCs 的 28.95.25%；G3 群细胞占骨髓总细胞的 6.041.55%，占骨髓MDSCs 的 20.954.98%； M1 群细胞占骨髓总细胞的 3.861.06%，占骨髓MDSCs 的 13.252.65%。 G1 群细胞体积较大主要是以环形核幼稚的粒细胞为主，G2 群细胞以环形核和分叶核的幼稚粒细胞为主， G3 群细胞是以分叶核和多形核幼稚粒细胞为主，而 M1 亚群细胞体积较小，胞质灰蓝色，多为圆形和椭圆形核，主要是幼稚单核细胞。G1 和 G2 亚群细胞均高表达 CD62L 和 Ly6C，M1和 G3 亚群细胞高表达 Ly6C 且部分高表达 CD62L，四个亚群细胞均低表达CD204 、CD206、TLR2 及共刺激分子 CD80、CD86 和 CD124（IL-4 受体） ，不表达 TLR4 和 CD210（IL-10 受体） 。 G2 和 G3 两群细胞分别可以不同程度的抑制 CD4+T（P0.05；P0.01 ）和 CD8+T（P0.01 ；P0.01）细胞增殖，而 G1和 M1 亚群细胞对 CD4+T 和 CD8+T 细胞增殖无明显抑制作用。G2 和 G3 两群细胞可能通过上调 IL-4、 IL-10 和 IFN- 等细胞因子的表达发挥免疫抑制作用（P0.01；P0.01） ，该免疫抑制作用可能与 Arg和 NOS2 两种酶的活性无关。6结论： 1. 正常小鼠骨髓初始 MDSCs（CD11b +细胞）能分别抑制 CD4+T 或 CD8+T 细胞增殖，并且能延缓和降低小鼠急性肝衰竭模型的死亡率。2. 根据初始MDSCs的CD11b 、Gr1 和Gfi1:GFP表达差异及细胞形态学差异可将其可分为M1、G1、G2 及G3 四个亚群：单核系亚群细胞M1（CD11b +Gr1+ GFP-）和粒系亚群细胞G1（CD11b lowGr1low GFP+） 、G2 （CD11b lowGr1high GFP+）及G3（CD11b highGr1high GFP+） 。3. 在骨髓初始 MDSCs 四个亚群中， G2 和 G3 均能不同程度的抑制 CD4+T 和CD8+T 细胞增殖，而 G1 和 M1 对 CD4+T 和 CD8+T 细胞的增殖无明显抑制作用；G2 亚群细胞可能通过分泌抗炎因子 IL-4、IL-10 发挥免疫抑制作用，G3 亚群细胞可能通过分泌抗炎因子 IL-10 和炎性因子 IFN- 发挥免疫抑制作用。4. 骨髓初始 MDSCs 的 G2 和 G3 亚群细胞对 CD4+T 和 CD8+T 细胞增殖抑制作用的机制可能与 NOS2 和 ArgI 两种酶无关。关键词：初始髓源抑制细胞；细胞亚群；免疫抑制；T 淋巴细胞7Identification of Bone Marrow Naive Myeloid-Derived Suppressor Cells Subpopulations with Distinct Phenotype and T Cell Suppressive ActivityABSTRACTObjectives：MDSCs are a group of heterogeneous cells, usually increased in tumor, infection, and inflammation and transplantation disease. It has a broad inhibitory function to immune system in pathological conditions and plays a regulator of the immune system; it is a debate about the inhibitory function of nave MDSCs in normal condittions. Currently, mixed-MDSCs or different subsets of MDSCs have been researched in different pathological conditions such as cancer and infection. This study used the bone marrow of Gfi1: GFP knockin mouse mice model, which isolated nave MDSCs, and sorted four subsets cells of naive MDSCs by FACS Aria, and using of modern immunological methods to understand the nave MDSCs and its four subsets of normal mice may have different phenotypes and T cell immune suppression; focus on different subsets of nave MDSCs inhibited T cells peroliferation and its mechanism. Methods：The bone marrow cells of C57BL/6J Gfi1: GFP knockin mouse model were separated and purificated, nave MDSCs of bone marrow were isolated by immunomagnetic beads (CD11b microbeads kit), the purity of nave MDSCs was detected by FCM; the morphology nave MDSCs was analyzed of by smear of centrifugation and staining of Wright-Giemsa; Identification nave MDSCs with CD4+T cells or CD8+T cells pre-stimulated by Dynabeads CD3/28 T-Activator and were cultured to detect CD4+ T and CD8+ T cells function by FCM. CD11blowGr1low 8GFP + (G1), CD11blowGr1highGFP+(G2), CD11bhighGr1highGFP+ (G3), CD11b+Gr1lowGFP- (M1) four subsets dependened on the differences of CD11b,Gr1 and Gfi1-GFP expression. Identification and isolation of purified four subsets function and identified the phenotype and analysed morphology by smear of centrifugation and staining of Wright-Giemsa; respectively purified G1, G2, G3 and M1 subsets cells with CD4+T cells or CD8+T cells pre-stimulated by Dynabeads CD3/28 T-Activator and were cultured to detect CD4+ T and CD8+ T cells function by FCM. G1, G2,G3 and M1 subsets cells were isolated from the model mice bone marrow and detected mRNA expression in a vary of cytokines of nave MDSC subsets, which play a detail molecular mechanisms of immunosuppression. Furthermore，to investigate the status of ArgI and NOS2 in the mechanism of immunosuppression, blocked ArgI ,NOS, and NOS2 respectly by L-NMMA, L-NIL and nor-NOHA and detected by FCM.Results：Nave MDSCs are important components of normal mice immune system, the morphology of total MDSCs in the bone marrow is large and small, the cytoplasm is blue, and the shape of nucleus is sub-leaf, ring and oval. CD4+T or CD8+T cells proliferation were inhibited by nave MDSCs (CD11b+ cells) of normal mice bone marrow, and the mortality of acute hepatic failure (AHF) mice models were delayed and decreased by nave MDSCs. It could be devided into CD11blowGr1low GFP + (G1), CD11blowGr1highGFP+(G2), CD11bhighGr1highGFP+ (G3), CD11b+Gr1lowGFP- (M1) four subsets dependened on the differences of CD11b,Gr1 and Gfi1-GFP expression. Furthermore, G1 subsets of nave MDSCs are about 1.190.38% of bone marrow total cells, and about 4.040.9% of nave MDSCs. G2 subsets of nave MDSCs are about 8.492.69% of bone marrow total cells, and about 28.95.25% of nave MDSCs. G3 subsets of nave MDSCs are about 6.041.55% of bone marrow total cells, and about 20.954.98% of nave MDSCs. M1 subsets of nave MDSCs are about 3.861.06% of bone marrow total cells, and about 13.252.65% of nave MDSCs. The volume of G1 subset are huge and the nucleus of large group cells are ring shape , the nucleus of G2 subset cells are ring and sub-leaf shape, the nucleus of G3 subset cells are sub-leaf 9and polymorphonuclear shape, the nucleus of G3 subset cells are sub-leaf and polymorphonuclear shape, while the volume of M1 subsets are smaller, and most of cells are immature monocytes. The subsets of G1 and G2 high expression of CD62L and Ly6C , M1 and G3 high expression of Ly6C, and high expression of CD62L partially. CD204, CD206, TLR2 and CD80, CD86 (costimulatory molecules) and CD124 (receptor of IL-4) are low expression on four subsets of nave MDSCs, and not expression of TLR4 and CD210 (IL-10). G2 and G3 subsets of nave MDSCs inhibit CD4+T or CD8+T cell proliferation respectly at different levels; however, G1 and M1 subsets of nave MDSCs had no significant inhibitory effect to CD4+T cells or CD8+T cells. G2 and G3 subsets of nave MDSCs IL-4, IL-10, IL-13, IFN- and other cytokines were increased expression of G2 and G3 subsets of nave MDSCs. In addition, Argand NOS2 is maybe no significant relationship with the immunosuppressive effects.Conclutions：1. CD4+T or CD8+T cells proliferation were inhibited by nave MDSCs (CD11b+ cells) of normal mice bone marrow, and the mortality of acute hepatic failure (AHF) mice models were delayed and decrease