CAJ | 학술논문

目的：探讨巨噬细胞在脂多糖（ LPS）的持续刺激下产生免疫抑制后的表型变化及对T细胞影响的分子机制。方法：蔗糖密度梯度离心法从全血中分离人外周血单个核细胞，结合磁珠细胞分选技术分选出单核细胞，体外诱导单核细胞分化为巨噬细胞，以未处理和IFN－γ处理为对照，对LPS处理48 h的巨噬细胞进行形态学观察、细胞表面分子（ HLA－DR、CD14、CCR7、HLA－ABC及CD40）表达的检测和细胞因子（ IL－10、IL－12、IL－6及TNF－α）分泌水平的检测。同时将LPS诱导的巨噬细胞与CD3＋T细胞进行异体共培养，进一步观察巨噬细胞对T细胞增殖能力的影响。用实时荧光定量PCR验证Toll样受体4（ TLR4）信号通路中的非MyD88依赖型途径相关分子的表达水平。结果：LPS处理48 h的巨噬细胞，抗原递呈能力（ HLA－DR）下降，免疫抑制细胞因子IL－10升高，把LPS诱导的巨噬细胞与异体T细胞共培养6 d，其促进CD8＋T细胞增殖的能力较弱。实时荧光定量PCR结果显示LPS持续刺激下巨噬细胞的TRIF、IRF3和CIITA均呈下调状态。结论：持续LPS处理巨噬细胞48 h 后，巨噬细胞呈现一种免疫抑制的状态，且其刺激CD8＋T细胞增殖的能力减弱，这种状态与非MyD88依赖型TLR4信号通路受损有关。
목적：탐토거서세포재지다당（ LPS）적지속자격하산생면역억제후적표형변화급대T세포영향적분자궤제。방법：자당밀도제도리심법종전혈중분리인외주혈단개핵세포，결합자주세포분선기술분선출단핵세포，체외유도단핵세포분화위거서세포，이미처리화IFN－γ처리위대조，대LPS처리48 h적거서세포진행형태학관찰、세포표면분자（ HLA－DR、CD14、CCR7、HLA－ABC급CD40）표체적검측화세포인자（ IL－10、IL－12、IL－6급TNF－α）분비수평적검측。동시장LPS유도적거서세포여CD3＋T세포진행이체공배양，진일보관찰거서세포대T세포증식능력적영향。용실시형광정량PCR험증Toll양수체4（ TLR4）신호통로중적비MyD88의뢰형도경상관분자적표체수평。결과：LPS처리48 h적거서세포，항원체정능력（ HLA－DR）하강，면역억제세포인자IL－10승고，파LPS유도적거서세포여이체T세포공배양6 d，기촉진CD8＋T세포증식적능력교약。실시형광정량PCR결과현시LPS지속자격하거서세포적TRIF、IRF3화CIITA균정하조상태。결론：지속LPS처리거서세포48 h 후，거서세포정현일충면역억제적상태，차기자격CD8＋T세포증식적능력감약，저충상태여비MyD88의뢰형TLR4신호통로수손유관。
[ ABSTRACT] AIM:To investigate the molecular mechanism and the immunosuppressive phenotype of macropha-ges under long-term exposure to lipopolysaccharide ( LPS) .METHODS:We used Ficoll-Hypaque density gradient centri-fugation combined with MicroBeads Separation Kits to separate peripheral blood mononuclear cells from human blood, and then induced the monocytes into macrophages.We observed the morphology of the macrophages by treating the cells with LPS for 48 h, in comparison with a negative control and IFN-γtreatment.ELISA was used to detect the levels of cytokines, such as IL-10, IL-12, IL-6 and TNF-α, and flow cytometry was used to detect the expression of the surface molecules (HLA-DR, CD14, CCR7, HLA-ABC and CD40).To observe the effect of macrophage on T cell proliferation, co-culture experiment was carried out for 6 d.Real-time PCR was used to validate the expression levels of molecules related to MyD88-independent pathway in Toll-like receptor 4 ( TLR4) signal pathway.RESULTS:The antigen-presenting ability of the macrophages was reduced and the IL-10 expression level was increased after the cells were treated with LPS for 48 h. We observed a poor proliferative capacity of CD8 +T cells after co-culturing of LPS-induced macrophages with CD3+T cells for 6 d.The results of real-time PCR indicated that TRIF, IRF3 and CIITA were down-regulated in LPS-induced macropha-ges.CONCLUSION:We successfully established a macrophage model in vitro and observed that LPS-induced macropha-ges into an immunosuppressive phenotype with poor CD8 +T cell proliferative capacity, in which MyD88-independent TLR4 signaling pathway was impaired.