成人外周血来源的树突状细胞体外诱导方法
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摘要
目的:探讨从成人外周血中提取单核细胞经体外诱导为树突状细胞(DC)的方法。方法:采用密度梯度离心法和细胞贴壁法从成人新鲜血液中分离出树突状细胞的前体单核细胞,利用重组人白介素4(rh IL-4)重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)经体外诱导5 d后,加入浓度为75 ng/m L的重组人肿瘤坏死因子-a(TNF-a)培养2 d。观察细胞形态,并用流式细胞仪检测技术检测DC表面分子标志物CD80-FITC、CD83-PE和HLA-DR-APC的表达。结果:沉降6 h后洗去淋巴细胞后的DC细胞呈圆形,大小均一,表面较光滑,贴壁生长。诱导第3天,DC细胞形态明显改变,体积变大,单个细胞开始呈簇状集落生长,部分细胞呈半悬浮状态。第5天细胞集落现象明显。加入rh TNF-a后,诱导第7天DC细胞悬浮,单个散开。成熟DC细胞CD80-FITC、CD83-PE、HLA-DRAPC表达率分别为81.59%、67.37%、85.38%。结论:密度梯度离心和细胞贴壁法可从外周血可获得DC的前体单核细胞,通过rh IL-4、rhGM-CSF和rh TNF-a联合诱导可得到成熟的DCs。
Objective:To investigate methods for extracting monocytes from adult peripheral blood and inducing them into dendritic cells(DC) in vitro.Methods:The dendritic cell precursor monocytes were isolated from the adult fresh blood by density gradient centrifugation and cell attachment,and recombinant human interleukin-4(rh IL-4) was used to recombine human granulocyte-macrophage colony-stimulating factor(GM-CSF).After 5 days of in vitro induction,75 ng/mL recombinant human tumor necrosis factor-a(TNF-a) was added and incubated for 2 days.Cell morphology was observed and the expressions of DC surface molecular markers CD80-FITC,CD83-PE and HLA-DR-APC was detected by flow cytometry.Results:After 6 hours of sedimentation,the DC cells after washing away lymphocytes were round,uniform in size,smooth in surface and adherent.On the third day of induction,the morphology of DC cells changed significantly,and the volume became larger.The individual cells began to grow into clusters,and some cells were semi-suspended.On the fifth day of induction,the cell colony was obvious.After the addition of rh TNF-a,on the seventh day of induction,the DC cells were suspended and spread individually.The expression rates of CD80-FITC,CD83-PE and HLA-DRAPC in mature DC cells were 81.59%,67.37% and 85.38%,separately.Conclusions:Density gradient centrifugation and cell attachment method can used to obtain the DC precursor progenitor cells from peripheral blood,and mature DCs can be obtained by the combined induction of rh IL-4,rh GM-CSF and rh TNF-a.
Objective:To investigate methods for extracting monocytes from adult peripheral blood and inducing them into dendritic cells(DC) in vitro.Methods:The dendritic cell precursor monocytes were isolated from the adult fresh blood by density gradient centrifugation and cell attachment,and recombinant human interleukin-4(rh IL-4) was used to recombine human granulocyte-macrophage colony-stimulating factor(GM-CSF).After 5 days of in vitro induction,75 ng/mL recombinant human tumor necrosis factor-a(TNF-a) was added and incubated for 2 days.Cell morphology was observed and the expressions of DC surface molecular markers CD80-FITC,CD83-PE and HLA-DR-APC was detected by flow cytometry.Results:After 6 hours of sedimentation,the DC cells after washing away lymphocytes were round,uniform in size,smooth in surface and adherent.On the third day of induction,the morphology of DC cells changed significantly,and the volume became larger.The individual cells began to grow into clusters,and some cells were semi-suspended.On the fifth day of induction,the cell colony was obvious.After the addition of rh TNF-a,on the seventh day of induction,the DC cells were suspended and spread individually.The expression rates of CD80-FITC,CD83-PE and HLA-DRAPC in mature DC cells were 81.59%,67.37% and 85.38%,separately.Conclusions:Density gradient centrifugation and cell attachment method can used to obtain the DC precursor progenitor cells from peripheral blood,and mature DCs can be obtained by the combined induction of rh IL-4,rh GM-CSF and rh TNF-a.
引文
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[2]Collin M,Bigley V.Human dendritic cell subsets:an update[J].Immunology,2018,154(1):3-20.
[3]van Dinther D,Stolk DA,van de Ven R,et al.Targeting C-type lectin receptors:a high-carbohydrate diet for dendritic cells to improve cancer vaccines[J].J Leukocyte Biol,2017,102(4):1017-1034.
[4]Wacleche VS,Tremblay CL,Routy JP,el at.The biology of monocytes and dendritic cells:contribution to HIV pathogenesis[J].Viruses,2018,10(2):65.
[5]吴耀松,尹素改,任闪闪.六君子汤对食管癌EC-9706细胞株树突状细胞成熟的影响[J].中医杂志,2018,59(6):508-512.
[6]杨姝瑞,刘嘉颖,刘建民,等.电针对SAMP8小鼠外周血树突状细胞CD40、CD80分子表达的影响[J].湖北中西药大学学报,2018,20(1):34-38.
[7]郑云梅,和晨辰,王世忠,等.树突状细胞与黑色素瘤细胞体外共培养体系对小鼠黑色素瘤形成的影响[J].山东医药,2017,57(5):1-3.
[8]Schülke S.Induction of interleukin-10 producing dendritic cells as a tool to suppress allergen-specific T helper 2 responses[J].Front Immuno,2018,9:455.
[9]Serrano I,Luque A,Aran JM.Exploring the immunomodulatory moonlighting activities of acute phase proteins for tolerogenic dendritic cell generation[J].Front Immuno,2018,9:892.