吞噬细胞的磷脂酰丝氨酸外翻与其血清依赖的调理作用的关系

Relationship between phosphatidylserine exposure and serum-dependent opsonization in phagocytes

目的:探讨外周血中单核吞噬细胞的磷脂酰丝氨酸(phosphatidylserine,PS)外翻与其血清依赖的调理作用之间的关系,为探索生理微环境对于吞噬细胞功能的影响奠定基础.方法:基于Ficoll-paque密度梯度离心法分离外周血单核细胞(peripheral blood mononuclear cells,PBMC),经过PBS充分洗涤之后利用含有和不含血清的RPMI 1640培养基处理细胞,一定时间之后利用Annexin V染色检测血清处理前后PS外翻的情况;通过比色法检测胞膜丙二醛(malondialdehyde,MDA)的含量,以考察脂质过氧化产物对Annexin V检测结果的影响;通过anti-CD3、anti-CD56、antiHLA-DR等不同的表面分子抗体染色,利用流式分析PBMC中不同细胞群的PS外翻情况;利用表达绿色荧光蛋白的细菌与PBMC共孵育,通过流式细胞术检测细胞的吞噬能力.结果:流式分析表明PBMC经血清处理后PS+细胞比例明显升高,并且这种升高发生在血清短暂的处理之后;血清处理对于细胞膜MDA的含量没有显著影响,排除了脂质过氧化对PS检测的影响;针对PBMC的细胞表型分析表明HLA-DR+单核吞噬细胞是PBMC中对血清处理敏感的主要细胞亚群,其在血清处理后Annexin V+细胞中的比例有显著的增加(P<0.05);细菌吞噬实验表明外翻的PS参与了血清诱导的调理作用.结论:吞噬细胞PS外翻是血清依赖的,并且这种外翻的PS参与了对细菌的调理作用.

AIM： To investigate the relationship between the exposure of phosphatidylserine（PS） and serum-dependent opsonization in peripheral blood mononuclear cells（PBMCs）, to lay a foundation for exploring the influence of physiological microenvironment on the function of phagocytes.MET HODS： PBMCs were isolated from the peripheral blood of healthy individuals using Ficoll-paque and treated by RPMI 1640 medium with or without serum after washing with PBS. The phosphatidylserine（PS） exposure was detected by annexin V staining before and after in vitro serum incubation. Malondialdehyde（MDA） in treated PBMCs was examined by spect rophotometry to evaluate the interference of lipid peroxidation. The PS exposure in different subsets of PBMCs was analyzed by flow cytometry with antiCD3, anti-CD56 and anti-HLA-DR staining. Phagocytosis was detected by FCM after incubation with bacteria expressing green fluorescent protein（GFP）. RESULTS： PS＋ cells significantly increased in PBMCs after serum incubation, and shortterm serum incubation immediately induced the increase of PS＋ cells. Serum incubation had no influence on the membrane MDAcontent, which excluded the interference by the products of lipid peroxidation. Cell subset analysis showed that HLA-DR＋ mononuclear phagocytes were the main cells exposing PS in PBMCs and significantly increased in Annexin V＋ cells. Bacterial phagocytosis showed that the exposed PS was involved in the serumdependent opsonization. CONCLUSION： The exposure of PS is serumdependent and is involved in the opsonization of bacteria by PBMCs.