Delirezh Nowruz, Majedi Leila, Asri Rezaei Siamak, Ranjkeshzadeh Hadi
Dept. of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran.
Dept. of Microbiology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Iran Biomed J. 2011;15(3):79-84.
Dendritic cells (DC) induce tumor or pathogen-specific T cell responses in humans. Several laboratories have developed culture systems, including maturation factors for human DC from peripheral blood monocytes. We comprehensively compared standard maturation stimulus, an autologous monocyte-conditioned medium (MCM), with heparin for their ability to promote uniformly mature DC that elicit T cell responses.
A short (4-day) priming of plastic adherent monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 with or without heparin was followed by 48-hour incubation in MCM to generate fully mature and stable DC. Phenotypic and functional analyses were carried out using anti-CD14 and anti-CD83 monoclonal antibodies, and mixed lymphocyte reaction, respectively.
We found that fully matured DC with a large amount of cytoplasm and copious dendritic projections were visible at the end of culturing period in the presence of MCM, heparin and MCM plus heparin. Thus, DC generated with these maturation factors are non-adherent and have typical satellite morphology. Flow cytometric analysis using anti-CD14 (monocyte marker) and anti-CD83 (mature DC marker) revealed that expression of CD14 decreased in MCM plus heparin-treated DC, and the expression of CD83 was increased when heparin and MCM used as a maturation factor. Functionally, MCM and MCM plus heparin-treated DC showed stronger mixed leukocyte reaction than heparin alone.
These results support the use of the MCM with heparin as maturation factor that could result in functionally mature monocyte-derived DC in comparison to either MCM or heparin alone.
树突状细胞（DC）可在人体内诱导肿瘤或病原体特异性T细胞反应。多个实验室已开发出培养系统，包括用于从外周血单核细胞生成人DC的成熟因子。我们全面比较了标准成熟刺激物、自体单核细胞条件培养基（MCM）和肝素促进能引发T细胞反应的均匀成熟DC的能力。
用粒细胞-巨噬细胞集落刺激因子（GM-CSF）和IL-4对塑料贴壁单核细胞进行短期（4天）预刺激，添加或不添加肝素，随后在MCM中孵育48小时以生成完全成熟且稳定的DC。分别使用抗CD14和抗CD83单克隆抗体进行表型和功能分析，并进行混合淋巴细胞反应。
我们发现，在存在MCM、肝素以及MCM加肝素的情况下，培养期结束时可见到具有大量细胞质和丰富树突状突起的完全成熟DC。因此，用这些成熟因子生成的DC不贴壁且具有典型的卫星形态。使用抗CD14（单核细胞标志物）和抗CD83（成熟DC标志物）进行的流式细胞术分析显示，在MCM加肝素处理的DC中CD14表达降低，而当使用肝素和MCM作为成熟因子时CD83表达增加。在功能上，MCM以及MCM加肝素处理的DC比单独使用肝素表现出更强的混合白细胞反应。
这些结果支持将MCM与肝素作为成熟因子使用，与单独使用MCM或肝素相比，这可能会产生功能成熟的单核细胞衍生DC。
