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目的探讨DC-SIGN的配体脂阿拉伯甘露聚糖(ManLAM)对肺结核患者树突状细胞黏附BCG水平的影响。方法构建带有EGFP基因的分枝杆菌穿梭质粒,电转化BCG,热诱导绿色荧光蛋白的表达,从而标记BCG;分离结核患者外周血单个核细胞,以GM-CSF和IL-4诱导树突状细胞生长,培养第5天用流式细胞术检测其表面DC-SIGN表达率,并在与不同浓度ManLAM预孵育后,与标记BCG结合,测定两者的黏附率。结果(1)分枝杆菌穿梭质粒构建成功,命名为pHSP70-EGFP。(2)用绿色荧光蛋白标记BCG成功。(3)肺结核患者外周血树突状细胞表面DC-SIGN表达率为(86.69±3.66)%。(4)伴随着ManLAM浓度的不断增加,肺结核患者外周血树突状细胞和BCG的黏附率不断下降。结论(1)可以用穿梭质粒pHSP70-EGFP在BCG中表达绿色荧光蛋白,从而标记BCG。(2)DC-SIGN分子是肺结核患者外周血树突状细胞表面结核分枝杆菌的重要受体。(3)纯化的ManLAM可以阻断肺结核患者外周血树突状细胞与BCG的黏附。
Objective To investigate the effect of DC-SIGN ligand Manila on the adhesion of dendritic cells to BCG in patients with pulmonary tuberculosis. Methods Mycobacterium shuttle plasmid with EGFP gene was constructed and electroporated into BCG. The expression of green fluorescent protein (GFP) was induced by heat-induced BCG. The peripheral blood mononuclear cells were isolated from patients with tuberculosis and the dendritic cells were induced by GM-CSF and IL-4 Cell growth was detected by flow cytometry. The expression of DC-SIGN on the surface was detected by flow cytometry. After preincubated with different concentrations of ManLAM, the cells were labeled with BCG and the adhesion rate was determined. Results (1) The mycobacterial shuttle plasmid was successfully constructed and named pHSP70-EGFP. (2) labeled with green fluorescent protein BCG success. (3) The expression of DC-SIGN on the surface of dendritic cells in patients with pulmonary tuberculosis was (86.69 ± 3.66)%. (4) With the continuous increase of ManLAM concentration, the adhesion rate of peripheral blood dendritic cells and BCG in patients with pulmonary tuberculosis decreased continuously. Conclusions (1) Green fluorescent protein can be expressed in BCG by shuttle plasmid pHSP70-EGFP to label BCG. (2) DC-SIGN is an important receptor of Mycobacterium tuberculosis on the surface of dendritic cells in patients with pulmonary tuberculosis. (3) The purified ManLAM can block the adhesion of peripheral blood dendritic cells and BCG in patients with pulmonary tuberculosis.