成人外周血来源树突状细胞体外诱导培养及成熟调控

人体内树突状细胞具有成熟和未知成熟两种形态。前者是早期免疫应答的强有力的抗原递呈细胞。后者具有截然不同的生物学特性，且能诱导免疫耐受。本研究从成人外周血中分离前体细胞，利用单核细胞条件性培养液，培养出比较均一的未成熟及成熟阶段的树突状细胞。第一阶段：从外周血分离出能粘附塑料的单核细胞，在GM－CSF＋IL-4存在条件下培养6－7d；第二阶段，加入单核细胞条件性培养液促进树突状细胞分化成熟，仅经第一阶段培养 的细胞主要为未成熟树突状细胞，仍然表达单核细胞的表达标志CD14，具有活跃的内,骐 MHC－II分子主要分布在胞内的MIIC器官；经第二阶段培养的树突状细胞则具有成熟树突状细胞的全部特征：典型的树突状形态，悬浮生长，MHC－II分子主要分布在包膜表面，．表达树突状细胞的特异性标志CD83，刺激同种T淋巴细胞增殖的能力强，并在脱离特定细胞因子环境3d后仍能保持该性状不变。由于该途径培养成熟、未成熟树突状细胞完全受外部因素调控，可能是获得均一的成熟和未成熟树突状细胞供科研和临床应用的较好途径。

Human Peripheral Blood Monocyte Derived Dendritic Cell Culture and Mature Regulation

Mature dendritic cells are potent antigen presenting cells that initiate primary immune responses, while immature dendritic cells have quite different properties from mature dendritic cells and are tolerance inducer actually. Here we describe the method of using monocyte condition medium to generate dendritic cells of different maturation phases from nonproliferating progenitors in human peripheral blood. The procedure involves two steps. The first step(or priming phase) is to work on a 6~7 day culture of plastic adherent blood monocyte in medium supplement with GM CSF and IL 4. The second step (or differentiation phase) requires the exposure to monocyte conditioned medium. Only the dendritic cells generated by the first step are actually immature, with strong immature dendritic cell features such as active endocytosis, the same expression of monocyte marker CD14, and much of the MHC class II still lies within intracellular compartments (MIIC). The second stage dendritic cells have all the features of mature dendritic cell, including a stellate shape, nonadherence to plastic, the expression of dendritic cells restricted marker CD83, and very strong T cell stimulatory function. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. Since progression from immature to mature dendritic cell is entirely dependent on exogenously added growth factor such as monocyte condition medium, the peripheral blood monocyte may help to harness synchronized population of mature and immature dendritic cells for studies or therapies.