Molecular and Medicine Research Center, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
Iran J Basic Med Sci. 2011 Jul;14(4):354-60.
Dendritic cells (DCs) are bone marrow-derived cells, which migrate to lymphoid and non-lymphoid organs via blood. Liver DCs are believed to play an important role in the regulation of hepatic allograft acceptance. However, because of inherent difficulties in isolating adequate numbers of DCs from liver, limited information is available on the phenotype and functions of liver DCs. To address this issue, we isolated DCs from normal C57BL/6 mouse liver using a modified procedure and described their immunophenotypic characteristics.
Non-parenchymal cells (NPCs) were obtained by collagenase digestion of perfused liver fragments and density gradient centrifugation (14.5% nycodenz column). After overnight (18 hr) incubation of the NPCs, enrichment for transiently adherent, low- density cells on 13% nycodenz gradients permitted the recovery of low numbers of cells (approximately 1.2-1.5 x 10(5) per liver), many of which displayed distinct DCs morphology (abundant cytoplasm with prominent projections and irregularly shaped nuclei).
Flowcytometric analysis revealed that most of these cells were recognized by anti-CD11c (60-70%). The results obtained from double staining with PE and FITC conjugated monoclonal antibodies indicated that these cells were CD11c(+)/MHC-II(+) (53%), CD11c(+)/CD86(+) (53.5%), CD11c(+)/ CD8α(+) (36%) and CD11c(+)/CD11b(+) (45%).
These findings indicate that the purity of DCs isolated by nycodenz gradient is higher than other reported methods. Considering the similar ratio of lymphoid (CD11c(+)/CD8α(+)) and myeloid (CD11c(+)/CD11b(+)) DCs in the liver, and the known role of lymphoid DCs in tolerance induction, it seems that this subpopulation of DCs is not the main reason of liver tolerogenecity. Therefore, other factors such as the immaturity of liver DCs or the effect of liver microenvironment on these cells, etc. may explain the acceptance of hepatic allograft.
树突状细胞（DCs）是骨髓来源的细胞，它们通过血液迁移到淋巴和非淋巴器官。肝 DCs 被认为在调节肝移植接受中起重要作用。然而，由于从肝中分离足够数量的 DCs 存在固有困难，因此关于肝 DCs 的表型和功能的信息有限。为了解决这个问题，我们使用改良的程序从正常 C57BL/6 鼠肝中分离 DCs，并描述了它们的免疫表型特征。
非实质细胞（NPCs）通过胶原酶消化灌注的肝碎片和密度梯度离心（14.5% nycodenz 柱）获得。在 NPCs 孵育过夜（18 小时）后，在 13% nycodenz 梯度上短暂黏附、低密度细胞的富集允许回收少量细胞（每个肝约 1.2-1.5x10(5)个），其中许多细胞呈现出明显的 DCs 形态（丰富的细胞质，突出的突起和不规则形状的核）。
流式细胞术分析显示，这些细胞中的大多数被抗 CD11c（60-70%）识别。用 PE 和 FITC 缀合的单克隆抗体双重染色的结果表明，这些细胞是 CD11c(+)/MHC-II(+)（53%）、CD11c(+)/CD86(+)（53.5%）、CD11c(+)/CD8α(+)（36%）和 CD11c(+)/CD11b(+)（45%）。
这些发现表明，通过 nycodenz 梯度分离的 DCs 的纯度高于其他报道的方法。考虑到肝中淋巴样（CD11c(+)/CD8α(+））和髓样（CD11c(+)/CD11b(+））DCs 的相似比例，以及淋巴样 DCs 在诱导耐受中的已知作用，似乎这种 DCs 亚群不是肝耐受原性的主要原因。因此，其他因素，如肝 DCs 的不成熟或肝微环境对这些细胞的影响等，可能解释了肝移植的接受。
