人外周血树突状细胞的超微结构及其细胞化学特征

目的　观察体外富集的人外周血树突状细胞（ｄｅｎｄｒｉｔｉｃ ｃｅｌｌｓ，ＤＣ） 的光镜形态学，细胞超微结构及细胞化学特征。方法　取健康人新鲜外周抗凝血，采用淋巴细胞分离液分离单个核细胞进行培养，经细胞因子（ｒｈＧＭＣＳＦ，ｒｈＩＬ４ ，ｒｈＴＮＦα） 联合刺激培养９～１２ 天，富集含ＤＣ的细胞群，光镜形态学分辨ＤＣ形态及含量，用抗人血ＤＣ表面特异单克隆抗体（ｍＡｂ Ⅻ） 确认ＤＣ的细胞化学特征。结果　体外培养富集的人外周血ＤＣ群的ＤＣ含量达７０ ％ ～８０ ％ 。透射电镜观察表明，细胞因子诱导的人外周血ＤＣ群可见到两类ＤＣ亚群：一类可能来自ＤＣ前体细胞，其细胞表面有大量不规则树枝状突起（ 粗细不等） ，细胞核不规则，核仁小，细胞化学染色［髓过氧化物酶（ＰＯＸ） 、非特异性酯酶（ＮＳＥ）］ 均为阴性；另一类是由单核细胞分化来的ＤＣ，细胞形态不规则，细胞表面粗糙，有大量皱褶及较粗大树枝状突起，胞核常呈现肾形或马蹄形，核内异染色质边集。结论　细胞因子诱导的人外周血ＤＣ群存在多相性。

The cytologic ultrastructure and cytochemicalfeaturesof human dendritic cellsfrom peripheral blood

OBJECTIVE: To observe the cytomorphological, cellular ultrastructure and cytochemical features of the dendritic cell (DC) enriched populations in vitro. METHODS: Fresh peripheral blood from healthy volunteers was collected and mononuclear cells (PBMC) were isolated on lymphocyte separating solution. The PBMC were cultured in mediums containing rhGM-CSF, rhIL-4 and rhTNF-alpha 9-12 days to generate DC-enriched populations, the morphological features and numbers of DC were identified and counted respectively by light microscope (LM) and confirmed by immunofluorescence assay (IFA), the ultrastructure characteristics of the DC populations were observed by transmission electron microscope, and the cytochemical features of them were also examined. RESULTS: The percentage of DCs was 70%-80% in the DC-enriched populations as confirmed by IFA using mABXII. Transmission electron microscopy revealed that the DC populations contained two subsets of DC, one was considered as precursor cell derived DC(PC-DC), the cell surface of which had many irregular dendrites with a irregular nucleus, small nucleoli, numerous mitochondria, but scanty lysosomes, ribosome, rough endoplasmic reticulum and free polysomes. Cytochemical staining showed the DCs were both peroxidase (POX) and non-specific esterase (NSE) negative. The other was indentified as monocyte-derived DC(MO-DC), the features of which included: irregular shape, many ruffles and some rough lames on cell surfaces, nephritic or horseshoe nuclei with a thick rim of heterochromatin, numerous mitochondria, much more lysosomes, ribosomes, rough endoplasmic reticulum and Golgiosome. The cytochemical reactions indicated that most of them had POX-positive granules, and were NSE slightly positive. CONCLUSION: Cytokines in vitro induced DCs from human peripheral blood are heterogeneous.