肺肿瘤小鼠MDSC、Treg及传统T细胞变化研究
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摘要
目的探讨肺肿瘤小鼠骨髓源性抑制细胞(MDSC)、调节性T细胞(Treg)和传统T细胞的变化及机制。方法采用配对设计将20只C57BL/6小鼠随机均分为Lewis肺癌细胞注射组(LLC组)和正常对照组(NC组),LLC组采用皮下注射LLC细胞100μL(1×10~6)制备肺肿瘤小鼠模型,对照组注射等量生理盐水。待肿瘤形成后取小鼠脾细胞,采用流式细胞仪检测肺肿瘤小鼠MDSC、Treg及CD4~+和CD8~+T细胞比例和数量变化,膜联蛋白-V(Annexin-Ⅴ)染色检测CD4~+和CD8~+T细胞凋亡变化,5-溴脱氧尿嘧啶核苷(BrdU)染色检测CD4~+和CD8~+T细胞增殖变化。结果与NC组相比,LLC组脾脏MDSC比例和数量明显增加,CD4~+Foxp3~+Treg所占CD4~+T细胞比例和数量明显增加,而CD4~+和CD8~+T细胞所占脾细胞比例和数量明显降低(均P<0.05)。与NC组相比,LLC组CD4~+和CD8~+T细胞增殖明显降低,同时CD8~+T细胞凋亡明显增加(P<0.05)。结论 MDSC和Treg细胞在肺肿瘤小鼠数量增加,同时,MDSC和Treg抑制CD4~+和CD8~+T细胞增殖,并促进CD8~+T细胞凋亡。
Objective To explore changes of the myeloid derived suppressor cell(MDSC), regulatory T cell(Treg),traditional T cell, and their mechanisms in lung tumor mice. Methods Twenty C57BL/6 mice were randomly divided intothe experimental and the normal control groups. The experimental group was injected with Lewis lung cancer cells(LLC, 100μL 1×106) subcutaneously to prepare the lung tumor model mice, the normal control group was given the same amount ofsaline(NC). Spleen cells were obtained from LLC and NC groups. Flow cytometry was used to detect the ratio and numberchanges of MDSC, Treg, CD4~+and CD8~+T cells in the lung tumor of mice. CD4~+and CD8~+T cell apoptosis were detected byAnnexin-Ⅴstaining, and their proliferation were detected by 5-bromine deoxidization uracil nucleoside(BrdU) incorporation.Results Compared with normal control mice, the ratio and number of MDSC in spleen increased significantly in LLC group(P<0.01), in addition, the ratio of CD4~+Foxp3~+Treg in CD4~+T cells and their number in spleen increased significantly in LLCgroup. However, the ratio and number of CD4~+and CD8~+T cells in spleen decreased significantly in LLC group(P<0.05).The proliferation of CD4~+and CD8~+T cells decreased significantly in LLC group compared with that of NC group(P<0.05),while the apoptosis of CD8~+T cells increased significantly(P<0.05). Conclusion MDSC and Treg cells increase in lungtumor model mice, which inhibit proliferation of CD4~+and CD8~+T cells and promote apoptosis of CD8~+T cells.
Objective To explore changes of the myeloid derived suppressor cell(MDSC), regulatory T cell(Treg),traditional T cell, and their mechanisms in lung tumor mice. Methods Twenty C57BL/6 mice were randomly divided intothe experimental and the normal control groups. The experimental group was injected with Lewis lung cancer cells(LLC, 100μL 1×106) subcutaneously to prepare the lung tumor model mice, the normal control group was given the same amount ofsaline(NC). Spleen cells were obtained from LLC and NC groups. Flow cytometry was used to detect the ratio and numberchanges of MDSC, Treg, CD4~+and CD8~+T cells in the lung tumor of mice. CD4~+and CD8~+T cell apoptosis were detected byAnnexin-Ⅴstaining, and their proliferation were detected by 5-bromine deoxidization uracil nucleoside(BrdU) incorporation.Results Compared with normal control mice, the ratio and number of MDSC in spleen increased significantly in LLC group(P<0.01), in addition, the ratio of CD4~+Foxp3~+Treg in CD4~+T cells and their number in spleen increased significantly in LLCgroup. However, the ratio and number of CD4~+and CD8~+T cells in spleen decreased significantly in LLC group(P<0.05).The proliferation of CD4~+and CD8~+T cells decreased significantly in LLC group compared with that of NC group(P<0.05),while the apoptosis of CD8~+T cells increased significantly(P<0.05). Conclusion MDSC and Treg cells increase in lungtumor model mice, which inhibit proliferation of CD4~+and CD8~+T cells and promote apoptosis of CD8~+T cells.
引文
[1]Alizadeh D,Larmonier N.Chemotherapeutic targeting of cancer-induced immunosuppressive cells[J].Cancer Res,2014,74(10):2663-2668.doi:10.1158/0008-5472.CAN-14-0301.
[2]Motz GT,Coukos G.Deciphering and reversing tumor immunesuppression[J].Immunity,2013,39(1):61-73.doi:10.1016/j.immuni.2013.07.005.
[3]Hoepner S,Loh JM,Riccadonna C,et al.Synergy between CD8 TCells and Th1 or Th2 Polarised CD4 T Cells for adoptiveimmunotherapy of brain tumours[J].PLoS One,2013,8(5):e63933.doi:10.1371/journal.pone.0063933.
[4]Diaz-Montero CM,Finke J,Montero AJ.Myeloid-derived suppressorcells in cancer:therapeutic,predictive,and prognostic implications[J].Semin Oncol,2014,41(2):174-184.doi:10.1053/j.seminoncol.2014.02.003.
[5]Lindau D,Gielen P,Kroesen M,et al.The immunosuppressivetumour network:myeloid-derived suppressor cells,regulatory Tcells and natural killer T cells[J].Immunology,2013,138(2):105-115.doi:10.1111/imm.12036.
[6]Yan YJ,He XH.Review the function and structure of interleukin-35 and its function in mediating treg cell immuno-suppression[J].Tianjin Med J,2014,42(12):1243-1245.[闫永嘉,何向辉.IL-35结构功能及其调控Treg细胞免疫抑制功能的研究进展[J].天津医药,2014,42(12):1243-1245].doi:10.3969/j.issn.0253-9896.2014.12.026.
[7]Movahedi K,Guilliams M,Van den Bossche J,et al.Identificationof discrete tumor-induced myeloid-derived suppressor cellsubpopulations with distinct T cell-suppressive activity[J].Blood,2008,111(8):4233-4244.doi:10.1182/blood-2007-07-099226.
[8]Youn JI,Nagaraj S,Collazo M,et al.Subsets of myeloid-derivedsuppressor cells in tumor-bearing mice[J].J Immunol,2008,181(8):5791-5802.doi:10.4049/jimmunol.181.8.5791.
[9]Zheng QH,Liu YW,Zhang XM.Changes of myeloid derived suppressorcells and T cells in lung cancer mice[J].Chinese Journal ofImmunology,2015,31(5):595-599.[郑全辉,刘英文,张雪梅.肺癌小鼠MDSC和T细胞变化[J].中国免疫学杂志,2015,31(5):595-599].doi:10.3969/j.issn.1000-484X.20 15.05.004.
[10]Li YD,Liu RQ,Liu J,et al.The proportion and significance of MDSC in the PBL of non-small cell lung cancer patients[J].Chinese Journal of Practical Medicine,2013,40(1):61-64.[李亚东,刘瑞青,刘杰,等.非小细胞肺癌患者外周血MDSC的比例及意义[J].中国实用医刊,2013,40(1):61-64].doi:10.3760/cma.j.issn.1674-4756.2013.01.026
[11]Fan HT,Lin HS,Li J,et al.The changes of CD4+CD25+Foxp3+regulatory T cells and myeloid-derived suppressor cells in Lewislung carcinoma-bearing mice and its relationship with tumor growth[J].Current Immunology,2009,29(5):370-375.[樊慧婷,林洪生,李杰,等.Lewis肺癌荷瘤小鼠CD4+CD25+Foxp3+调节性T细胞及髓样抑制细胞与肿瘤生长的关系[J].现代免疫学,2009,29(5):370-375].
[12]Sawant DV,Vignali DA.Once a Treg,always a Treg?[J].ImmunolRev,2014,259(1):173-191.doi:10.1111/imr.12173.
[13]Waldron TJ,Quatromoni JG,Karakasheva TA,et al.Myeloidderived suppressor cells:Targets for therapy[J].Oncoimmunology,2013,2(4):e24117.doi:10.4161/onci.24117.
[14]Rodriguez PC,Quiceno DG,Ochoa AC.L-arginine availabilityregulates T-lymphocyte cell-cycle progression[J].Blood,2007,109(4):1568-1573.doi:10.1182/blood-2006-06-031856.
[15]Daigneault M,De Silva TI,Bewley MA,et al.Monocytes regulatethe mechanism of T-cell death by inducing Fas-mediated apoptosisduring bacterial infection[J].PLo S Pathog,2012,8(7):e1002814.doi:10.1371/journal.ppat.1002814.
[16]Guo CL,Yang XH,Cheng W,et al.Expression of Fas/Fas L inCD8+T and CD3+Foxp3+Treg cells--relationship with apoptosisof circulating CD8+T cells in hepatocellular carcinoma patients[J].Asian Pac J Cancer Prev,2014,15(6):2613-2618.
[2]Motz GT,Coukos G.Deciphering and reversing tumor immunesuppression[J].Immunity,2013,39(1):61-73.doi:10.1016/j.immuni.2013.07.005.
[3]Hoepner S,Loh JM,Riccadonna C,et al.Synergy between CD8 TCells and Th1 or Th2 Polarised CD4 T Cells for adoptiveimmunotherapy of brain tumours[J].PLoS One,2013,8(5):e63933.doi:10.1371/journal.pone.0063933.
[4]Diaz-Montero CM,Finke J,Montero AJ.Myeloid-derived suppressorcells in cancer:therapeutic,predictive,and prognostic implications[J].Semin Oncol,2014,41(2):174-184.doi:10.1053/j.seminoncol.2014.02.003.
[5]Lindau D,Gielen P,Kroesen M,et al.The immunosuppressivetumour network:myeloid-derived suppressor cells,regulatory Tcells and natural killer T cells[J].Immunology,2013,138(2):105-115.doi:10.1111/imm.12036.
[6]Yan YJ,He XH.Review the function and structure of interleukin-35 and its function in mediating treg cell immuno-suppression[J].Tianjin Med J,2014,42(12):1243-1245.[闫永嘉,何向辉.IL-35结构功能及其调控Treg细胞免疫抑制功能的研究进展[J].天津医药,2014,42(12):1243-1245].doi:10.3969/j.issn.0253-9896.2014.12.026.
[7]Movahedi K,Guilliams M,Van den Bossche J,et al.Identificationof discrete tumor-induced myeloid-derived suppressor cellsubpopulations with distinct T cell-suppressive activity[J].Blood,2008,111(8):4233-4244.doi:10.1182/blood-2007-07-099226.
[8]Youn JI,Nagaraj S,Collazo M,et al.Subsets of myeloid-derivedsuppressor cells in tumor-bearing mice[J].J Immunol,2008,181(8):5791-5802.doi:10.4049/jimmunol.181.8.5791.
[9]Zheng QH,Liu YW,Zhang XM.Changes of myeloid derived suppressorcells and T cells in lung cancer mice[J].Chinese Journal ofImmunology,2015,31(5):595-599.[郑全辉,刘英文,张雪梅.肺癌小鼠MDSC和T细胞变化[J].中国免疫学杂志,2015,31(5):595-599].doi:10.3969/j.issn.1000-484X.20 15.05.004.
[10]Li YD,Liu RQ,Liu J,et al.The proportion and significance of MDSC in the PBL of non-small cell lung cancer patients[J].Chinese Journal of Practical Medicine,2013,40(1):61-64.[李亚东,刘瑞青,刘杰,等.非小细胞肺癌患者外周血MDSC的比例及意义[J].中国实用医刊,2013,40(1):61-64].doi:10.3760/cma.j.issn.1674-4756.2013.01.026
[11]Fan HT,Lin HS,Li J,et al.The changes of CD4+CD25+Foxp3+regulatory T cells and myeloid-derived suppressor cells in Lewislung carcinoma-bearing mice and its relationship with tumor growth[J].Current Immunology,2009,29(5):370-375.[樊慧婷,林洪生,李杰,等.Lewis肺癌荷瘤小鼠CD4+CD25+Foxp3+调节性T细胞及髓样抑制细胞与肿瘤生长的关系[J].现代免疫学,2009,29(5):370-375].
[12]Sawant DV,Vignali DA.Once a Treg,always a Treg?[J].ImmunolRev,2014,259(1):173-191.doi:10.1111/imr.12173.
[13]Waldron TJ,Quatromoni JG,Karakasheva TA,et al.Myeloidderived suppressor cells:Targets for therapy[J].Oncoimmunology,2013,2(4):e24117.doi:10.4161/onci.24117.
[14]Rodriguez PC,Quiceno DG,Ochoa AC.L-arginine availabilityregulates T-lymphocyte cell-cycle progression[J].Blood,2007,109(4):1568-1573.doi:10.1182/blood-2006-06-031856.
[15]Daigneault M,De Silva TI,Bewley MA,et al.Monocytes regulatethe mechanism of T-cell death by inducing Fas-mediated apoptosisduring bacterial infection[J].PLo S Pathog,2012,8(7):e1002814.doi:10.1371/journal.ppat.1002814.
[16]Guo CL,Yang XH,Cheng W,et al.Expression of Fas/Fas L inCD8+T and CD3+Foxp3+Treg cells--relationship with apoptosisof circulating CD8+T cells in hepatocellular carcinoma patients[J].Asian Pac J Cancer Prev,2014,15(6):2613-2618.