人外周单个核细胞来源树突细胞成熟与肺炎支原体膜脂蛋白的影响

Influence of lipid associated membrane protein of mycoplasma pneumoniae on maturation of dendritic cells from human peripheral blood mononuclear cells

目的:实验着眼于肺炎支原体膜脂蛋白对人外周血单个核细胞来源的树突细胞的表型(CD83,CD86)及分泌细胞因子的作用,探讨肺炎支原体加重哮喘的机制是否是改变了树突细胞的性状。方法:①对象:所有外周血采自正常人,志愿献血者为武汉大学医学院2005级硕士博士研究生6人;实验用肺炎支原体膜脂蛋白购自广州华银医药科技有限公司。②实验过程及分组:从肘静脉采集志愿者20mL新鲜全血,以不连续密度梯度离心法及贴壁法获取单核细胞,加入重组人粒细胞-巨噬细胞集落刺激因子,重组人白细胞介素4培养5d得到不成熟树突细胞后,分别予肺炎支原体膜脂蛋白,脂多糖和空白刺激再培养2d。③评估:用流式细胞仪检测各组树突细胞表面表达CD83,CD86的情况,用酶联免疫吸附法检测各组树突细胞分泌白细胞介素12的水平。结果:①肺炎支原体膜脂蛋白组树突细胞表达CD83,CD86高于未刺激组(P<0.01,P<0.01);脂多糖组树突细胞表达CD83,CD86高于未刺激组(P<0.01,P<0.01);肺炎支原体膜脂蛋白组树突细胞表达CD83较脂多糖组无差异,表达CD86高于脂多糖组(P<0.05)。②肺炎支原体膜脂蛋白组树突细胞分泌白细胞介素12高于未刺激组(P<0.01);脂多糖组树突细胞分泌白细胞介素12高于未刺激组(P<0.01);肺炎支原体膜脂蛋白组树突细胞分泌白细胞介素12低于脂多糖组(P<0.05)。结论:肺炎支原体膜脂蛋白可刺激未成熟树突细胞分化成熟,但较脂多糖刺激的不同,前者刺激成熟的树突细胞在呈递抗原给T细胞时,可进一步使T细胞向Th2极化,导致Th1/Th2平衡失调,从而加重哮喘。

AIM： The study explored the expression of the phenotype （CD83, CD86） and cytokine profile of human peripheral blood mononuclear cell （PBMC）-derived dendritic cells （DCs） stimulated by the lipid associated membrane protein of mycoplasma pneumoniae （MP-LAMP）, and investigated whether the changes in DC character is one of the mechanism by which MP infection exacerbate asthma. METHODS： ①Fresh peripheral blood was gathered from six volunteers who were master or doctor from Medical College of Wuhan University in 2005. Lipid associated membrane protein of mycoplasma pneumoniae was purchased from Guangzhou Huayin Medical Technology Co., Ltd. ②Fresh whole blood （20 mL） was collected from volunteer ulnar vein. PBMC was obtained by discontinuous density gradient centrifugation and monocyte was gained by adherence method and cultured with recombinant human granulocyte-macrophage colony stimulating factor （rhGM-CSF） and recombinant human interleukin （rhIL-4） as immature DC （iDC）. Then the iDC were divided to three groups that one was stimulated by MP-LAMP, another by lipopolysaccharide （LPS）, and the third as control for 2 days. ③Flow cytometer was used to detect the expression of phenotype on DC, and enzyme linked immunosorbent assay （ELISA） method was used to detect the IL-12 of cytokine by DC. RESULTS： ①The expressions of CD83 and CD86 on DCs stimulated by MP-LAMP were remarkably higher than non-stimulation group （P 〈 0.01, P 〈 0.01）. The expressions of CD83 and CD86 on DCs stimulated by LPS were remarkably higher than non-stimulation group （P 〈 0.0 1, P 〈 0.01）. The expression of CD83 on DCs stimulated by MP-LAMP was as the same as LPS group, but DC86 expression was higher than LPS group （P 〈 0.05）. ②IL-12 secreted by DCs in MP-LAMP group was higher than non-stimulation group （P 〈 0.01）. IL-12 secreted by DCs in LPS group was higher than non-stimulation group （P 〈 0.01）. IL-12 secreted by DCs in MP-LAMP group was lower than LPS group （P 〈 0.05）. CONCLUSION： MP-LAMP can induce iDC become mature, but the structure and function are different from DC stimulated by LPS. When the mature DC submit antigen to T cells, it can polarize T cells to Th2, and induce Th1/Th2 cytokine disequilibrium. Thus this kind of DC can exacerbate asthma.