小鼠骨髓DCs的高效扩增及其表型特征

Large-scale expansion and phenotype of mouse bone marrow DCs

目的:建立体外大量扩增骨髓树突状细胞(bone marrow derived dendritic cells,Bm-DCs)的方法,并分析其免疫表型特征。方法:以粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)、白细胞介素4(interleukin-4,IL-4)从C57BL/6小鼠骨髓细胞中诱导产生骨髓未成熟树突状细胞(immol/Lature DCs,imDCs),6 d后以脂多糖(lipopolysaccharide,LPS)进一步刺激产生成熟树突状细胞(mature DCs,mDCs)。以流式细胞技术检测imDCs、mDCs的CD11c、MHCⅡ、CD86、B220的表达。同时检测imDCs、mDCs刺激近交系BALB/c小鼠CD4+T淋巴细胞增殖的功能。结果:(1)每只小鼠的双侧股骨可以得到(4.0～6.0)×106个DCs;(2)CD11c、MHCⅡ、CD86、B220的表达率在imDCs和mDCs分别为(71.67±1.53)%、(25.67±2.08)%、(7.67±1.53)%、(9.00±1.00)%;(81.67±1.53)%、(55.67±3.06)%、(45.33±2.08)%、(8.00±1.00)%,其中imDCs、mDCs的CD11c、MHCⅡ、CD86的表达差异具有统计学意义(P<0.01);两组DCs的B220均为低表达;(3)成熟DCs具有很强的刺激近交系小鼠T淋巴细胞增殖的能力。结论:联合应用GM-CSF、IL-4可从小鼠骨髓中诱导扩增大量DCs;诱导产生的DCs的免疫表型特征为CD11chiMHC-Ⅱ+B220-,总体与常规DCs(conventional DCs,cDCs)相似。

Objective： To establish a method of large-scale expanding bone marrow derived dendritic cells（Bm-DCs） in vitro,and analysis its immol/Lune phenotype.Methods： Bone marrow cells were isolated from both femurs of murine,then cultured in medium supplemented with GM-CSF and IL-4.On days 7,the loosely adherent cells were collected as immol/Lature DCs（imDCs）.To get mature DC（mDCs）,imDCs on days 6 were stimulated by LPS for additional 16 hours,the expression of CD11c,MHCⅡ,CD86,B220 on imDCs and mDCs was detected by flow cytometry,and then examined the function of expanded DCs stimulating the proliferation of allogeneic CD4＋T lymphocytes.Results：（1）Each mouse bilateral femoral can get 4.50×106~6.50×106 DCs.（2）The expression of CD11c,MHCⅡ,CD86,B220 rate on imDCs and mDCs were（71.67±1.53）%,（25.67±2.08）%,（7.67±1.53）%,（9.00±1.00）%;（81.67±1.53）%,（55.67±3.06）%,（45.33±2.08）%,（8.00±1.00）%,respectively,the difference of the expression of CD11c,MHCⅡ,CD86 was statistically significant between imDCs and mDCs（P〈0.01）,while the expression of B220 was negative on both DCS.（3）The mature DC has stronger potential in stimulating T lymphocytes proliferation.Conclusion： Combined application of GM-CSF and IL-4,a large number of DCs can be generated from murine bone marrow,the immol/Lune phenotype of DCs induced by GM-CSF was CD11chi MHC-Ⅱ＋ B220-,similar to that of conventional DCs（cDCs）.