rmIL-4和rmGM-CSF联合应用诱导小鼠骨髓造血干细胞向树突状细胞分化的结果  

Results of combination of rmIL-4 and rmGM-CSF to induce differentiation of mouse bone marrow hematopoietic stem cells into DCs

作　　者：赵商岐 夏开德[2] 武娟 周彦霞 刘玉武 周文涛[3] 周晓涛 ZHAO Shangqi;XIA Kaide;WU Juan;ZHOU Yanxia;LIU Yuwu;ZHOU Wentao;ZHOU Xiaotao(Department of Immunology,Basic Medical College,Xinjiang Medical University,Urumqi 830011,China;不详)

机构地区：[1]新疆医科大学基础医学院免疫学教研室,乌鲁木齐830011 [2]贵阳市妇幼保健院 [3]新疆医科大学第五附属医院

出　　处：《山东医药》2021年第33期11-14,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81760656);新疆维吾尔自治区自然科学基金项目(2018D01C157)。

摘　　要：目的观察重组小鼠白细胞介素-4(rmIL-4)和重组小鼠粒细胞巨噬细胞集落刺激因子(rmGM-CSF)联合应用诱导小鼠骨髓造血干细胞向树突状细胞(DC)分化的结果。方法6~8周的C57小鼠,处死后取股骨和胫骨,取骨髓细胞,使用rmIL-4和rmGM-CSF进行诱导、培养。诱导第0、3、8、12天时,在倒置显微镜下观察细胞形态及生长状况。诱导第0、6、12天时,采用流式细胞术进行细胞表型鉴定(以CD45^(+)的细胞中CD11c^(+)CD86^(+)细胞占比表示)。诱导第6天时,收集细胞悬液后加入HIS-EgG1Y162抗原分别培养12 h与24 h后,采用流式细胞术进行细胞抗原捕获能力鉴定及成熟度观察(以结合HIS-EgG1Y162抗原的细胞占比表示细胞抗原捕获能力,以CD45^(+)的细胞中CD11c^(+)CD86^(+)、CD11c^(+)MHC-Ⅱ+细胞占比表示细胞成熟度)。结果诱导第0天时,细胞体积较小,呈类圆形悬浮于细胞培养液中;诱导第3天时,少量细胞开始出现“伪足”,细胞成团生长且数量开始增多;诱导第8天时,细胞体积增大,细胞出现明显的“突触”,呈多边形聚集于培养皿中央;诱导第12天时,细胞形态不规则,突触明显变长,集落细胞团形成。诱导第0、6、12天时,CD45^(+)的细胞中CD11c^(+)CD86^(+)细胞占比分别为1.170%±0.302%、6.120%±0.245%、16.000%±0.294%,两两比较,P均<0.05。诱导第6天时,加入HIS-EgG1Y162抗原共培养12 h后,结合HIS-EgG1Y162抗原的树突状细胞占比为1.760%±0.065%,CD45^(+)的细胞中CD11c^(+)CD86^(+)、CD11c^(+)MHC-Ⅱ+细胞占比分别为8.67%±0.38%、11.90%±0.49%。;加入HIS-EgG1Y162抗原共培养24 h后,结合HIS-EgG1Y162抗原的树突状细胞占比为13.400%±0.408%,CD45^(+)的细胞中CD11c^(+)CD86^(+)、CD11c^(+)MHC-Ⅱ+细胞占比分别为20.10%±0.29%、16.70%±0.24%;两者相比,P均<0.05。结论rmIL-4和rmGM-CSF联合应用诱导培养小鼠骨髓造血干细胞,可获得不同分化阶段的DC,且获得的未成熟DC具有较强的抗原捕获能力。Objective To observe the results of the combination of recombinant mouse interleukin-4(rmIL-4)and recombinant mouse granulocyte macrophage colony-stimulating factor(rmGM-CSF)to induce differentiation of mouse bone marrow hematopoietic stem cells into dendritic cells(DCs).Methods C57 mice at 6 to 8 weeks were executed and the femur and tibia bones were taken for bone marrow cells.RrmIL-4 and rmGM-CSF were used for induction and culture.On day 0,3,8 and 12 of induction,the cell morphology and growth were observed under inverted microscope.On day 0,6 and 12 of induction,cell phenotype was determined by flow cytometry(represented by the proportion of CD11c^(+)CD86^(+) cells in CD45^(+) cells).On the 6th day of induction,cell suspension was collected and HIS-EgG1Y162 antigen was added to culture for 12 h and 24 h,respectively.Flow cytometry was used to identify and observe the capture ability of cell antigen(the proportion of cells binding HIS-EgG1Y162 antigen indicated the capture ability of cell antigen,and the maturity was expressed by the proportion of CD11c^(+)CD86^(+) and CD11c^(+)MHC-Ⅱ^(+) cells in CD45^(+) cells).Results On the 0 d of induction,the cells were small in size and suspended in cell culture medium in a circular shape.On the 3rd day of induction,a small number of cells began to appear"pseudopodia",and the cells grew into clumps and the number began to increase.On the 8th day of induction,the cell volume increased and the cells appeared obvious"synapses",which were polygons clustered in the center of the petri dish.On the 12th day of induction,cell morphology was irregular,synapses became significantly longer,and colony cells formed.On the 0,6th and 12th days of induction,the proportion of CD11c^(+)CD86^(+) cells in CD45^(+) cells was 1.170%±0.302%,6.120%±0.245%,and 16.000%±0.294%,respectively(all P<0.05).On the 6th day of induction,the proportion of DCs combined with HIS-EgG1Y162 antigen was 1.760%±0.065%after 12 h co-culture with his-EgG1Y162 antigen.The proportion of CD11c^(+)CD86^(+) and CD

关 键 词：树突状细胞 造血干细胞 骨髓造血干细胞 抗原捕获 

分 类 号：R593[医药卫生—内科学]