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 den-dritic cells （DC） in vitro. Methods： The dendritic cell precursor monocytes were isolated from the adult fresh blood by density gradi-ent centrifugation and cell attachment, and recombinant human interleukin-4 （ rhIL-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 rhTNF-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 rhIL-4, rhGM-CSF and rhTNF-a.
作者
李金丽
王万聪
李冲慧
赵胜男
司远方
夏兴洲
LI Jinli;WANG Wancong;LI Chonghui;ZHAO Shengnan;SI Yuanfang;XIA Xingzhou(Department of Gastroenterology of the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou Henan 450000,Chin)
出处
《中国民康医学》
2018年第15期77-79,共3页
Medical Journal of Chinese People’s Health
关键词
密度梯度离心
细胞贴壁法
树突状细胞
表面抗原
诱导
Density gradient centrifugation
Cell attachment method
Dendritic cell
Surface antigen
Induction
