A Study of Antigen Presenting Dysfunction of Dendritic Cell from Peripheral Blood in the Patients with Connective Tissue Diseases

我们成功地从人外周血中制备了树突状细胞(DC)。总之，从50ml肝素化的外周血中用Ficoll-Hypaque梯度法获得单个核细胞，然后在培养皿中培养。去除未贴壁细胞，超高培养贴壁细胞。DC从碟子上分离出来。DC组分在抗人免疫球蛋白包被的塑料培养皿中培养，以排除巨噬细胞和B细胞。如果需要，加入抗3C10、Leul和Leul4+补体的单抗，以杀死每个污染细胞。DC具有特征性，其表面存在HLADR和C3biR，而Fc受体、表面Ig和OKT3抗原缺失。功能研究证实DC是混合淋巴细胞反应(MLR)的“活性刺激因子”。系统性红斑狼疮(SLE)患者的MLR明显降低。用MLR法检测SLE-DC功能。新的发现是SLE患者MLR降低的主要原因是SLE-DC功能受损，而不是几位作者以前报道的T细胞功能受损。DC与自体或异体T细胞形成特征性细胞聚集体(簇)。形成簇状的T细胞主要为OKT4阳性。集落培养上清液中含有SAC刺激的B细胞分化为抗体分泌细胞(BSF)的因子。由SLE-DC形成的集落培养上清液显示极低的BSF活性，表明SLE-DC存在功能异常。

We succeeded in preparation of dendritic cell(DC) from human peripheral blood. In brief, mononuclear cells were obtained by Ficoll-Hypaque gradients from 50ml heparinized peripheral blood, and then cultured in Petri dish. Non-adherent cells were removed and adherent cells were cultured overhight. The DC were detached from the dish. The DC fraction was cultured in anti-human IgG coated plastic dish for the purpose of excluding of macrophage and B cell. Monoclonal antibodies to 3C10, Leul and Leul4 plus complement were added for killing each contaminating cells if needed. DC possessed characteristic prosseses and on its surface HLA-DR and C3bireceptor were present, but Fc-receptor, surface Ig and OKT 3 antigen absent. Functional study proved that DC was "active stimulator" in mixed lymphocyte reaction (MLR). Reduced MLR was well documentated in the patients with systemic lupus erythematosus(SLE). We examined SLE-DC function using MLR assay. New findings were obtained that major cause of reduced MLR in the patients with SLE resulted in impaired function of SLE-DC and not in T cells as before reported by several authors. DC formed characteristic cell aggregates with autologous or allogneic T cells (cluster). T cells formed cluster were mainly OKT4 positive. Cultured cluster supernatant was proved to contain some factors to generate SAC-stimulated B cells into antibody secreting cells(BSF). Supernatant from cluster formed by SLE-DC showed very low grade BSF activities, indicating that SLE-DC had functional abnormalities.