少量人外周血体外培养鉴定单核细胞来源树突状细胞方法初探

Culture and identification of monocyte-derived dendritic cells from small volume of human peripheral blood in vitro

目的:在少量人外周血条件下体外培养并鉴定单核细胞来源树突状细胞(Monocyte-derived dendritic cells,Mo DC)。方法:取健康成人少量新鲜外周血经改良密度梯度离心法分离获得单核细胞,加入重组人粒-巨噬细胞集落刺激因子(rh GM-CSF)、重组人白细胞介素4(rh IL-4)诱导Mo DC生长,并用肿瘤坏死因子α(TNF-α)刺激成熟,倒置显微镜及扫描电镜观察细胞形态;分别于培养第4天和第7天用流式进行表型鉴定、CCK-8法检测同种异体混合淋巴细胞反应鉴定抗原递呈能力。结果:Mo DC呈类圆形,聚集成团,悬浮生长,扫描电镜观察其表面有典型毛刺状突起;流式检测Mo DC高表达CD11c、CD1a,经TNF-α刺激后的Mo DC表面MHCⅡ、CD80、CD83、CD86表达均较培养4 d的Mo DC明显升高,具有统计学意义(P<0.05);经TNF-α刺激后的Mo DC刺激同种异体淋巴细胞增殖能力较培养4 d的Mo DC明显升高,具有统计学意义(P<0.05)。结论:用本实验方法可从少量人外周血中获得成熟Mo DC,为其在多种变态反应疾病、自身免疫性疾病及肿瘤疫苗等领域的研究提供基础保障。

Objective： To culture and identify monocyte-derived dendritic cells （MoDC） from small volume of human peripheral blood in vitro. Methods： Monocytes were obtained from healthy human fresh peripheral blood by improved ficoll density gradient centrifugation. Human recombinant granuloeyte-macrophage colony--stimulating factor （rhGM-CSF） and human recombinant interleukin-4 （rhlL4） were added to induce the production of MoDC, tumor necrosis factor-or （TNF-ot）was added to stimulate matura- tion. Then the morphological features were observed by invert optical microscope and scanning electron microscope, the phenotypes were measured by flow cytometry and allogeneic mixed lymphocyte reaction were measured by CCK-8 to identify the function of MoDC to stimulate the proliferation of lymphoeytes on the fourth day and the seventh day respectively. Results： MoDC were suspension and cluster growth with a quasi-circular shape. The dendritic synapses, the typical morphological feature of MoDC, could be observed by scanning electron microscope;there were high levels of CD11 c/CD1 a on MoDC surface, the levels of MHC I1/CD80/CD83/CD86 were significant higher through the stimulation of TNF-α（ P〈0, 05 ）, Also, the ability to stimulate the proliferation of allogeneic lymphocytes was significant stronger （ P〈0.05 ）. Conclusion： Mature MoDC can be obtained from small volume of human peripheral blood in vitro by the method which provides the basis for further studying the effects of MoDC in autoimmune diseases, allergic diseases and tumor vaccines.