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。简单地说，从50ml肝素化的外周血中通过Ficoll-Hypaque梯度获得单个核细胞，然后在培养皿中培养。非贴壁细胞去除，贴壁细胞过夜培养。DC从碟子上分离出来。DC部分在抗人IgG包被塑料培养皿中培养，目的是排除巨噬细胞和B细胞。如有需要，加入3C10、Leul和Leul4 +补体单克隆抗体杀死每一个污染细胞。DC具有特异性，其表面存在HLA-DR和c3双受体，但不存在fc受体、表面Ig和okt3抗原。功能研究证实DC在混合淋巴细胞反应（MLR）中是“活性刺激物”。系统性红斑狼疮（SLE）患者MLR降低有充分的文献记载。我们使用MLR法检测slel - dc功能。新的研究发现，SLE患者MLR降低的主要原因是SLE- dc功能受损，而不是像之前几位作者报道的那样是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）。形成簇的T细胞以OKT4阳性为主。培养的簇状上清含有使sac刺激的B细胞转化为抗体分泌细胞（BSF）的因子。SLE-DC聚集形成的上清显示极低的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.