CAJ | 학술논문

目的寻找人髓系细胞触发受体-1(TREM-1)的天然配体,为阐明脓毒症的发病机制提供理论依据.方法分离人外周血中性粒细胞和单核细胞进行原代培养.在两种分离培养的细胞中分别加入热失活的金黄色葡萄球菌、铜绿假单胞菌、结核分枝杆菌或高渗诱导金黄色葡萄球菌L型、铜绿假单胞菌L型处理24 h.在两种分离培养的细胞中分别加入超声提取的金黄色葡萄球菌细胞壁、铜绿假单胞菌细胞壁或结核分枝杆菌细胞壁处理24 h.在两种分离培养的细胞中分别加入各细胞壁三大组分(细胞壁多糖、细胞壁脂类、细胞壁蛋白)处理24 h.用荧光定量聚合酶链反应(PCR)检测各处理组细胞TREM-1 mRNA水平,用酶联免疫吸附法(ELISA)检测各处理组细胞培养上清液中肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)浓度.结果金黄色葡萄球菌和铜绿假单胞菌的菌体、细胞壁和细胞壁多糖处理后中性粒细胞和单核细胞TREM-1mRNA水平以及细胞培养上清液中TNF-α和IL-1β浓度均明显升高.与空白对照组相比,金黄色葡萄球菌细胞壁多糖处理后中性粒细胞TREM-1 mRNA水平增加了(3.86±0.20)倍,单核细胞TREM-1 mRNA水平增加了(5.15±0.56)倍(均P＜0.05);铜绿假单胞菌细胞壁多糖处理后中性粒细胞TREM-1 mRNA水平增加了(4.03±0.15)倍,单核细胞TREM-1 mRNA水平增加了(7.22±0.73)倍(均P＜0.05).加入能竞争性结合TREM-1配体的LP17可以削弱此效应;而结核分枝杆菌的菌体、细胞壁和细胞壁组分均无此效应.结论人TREM-1天然配体位于金黄色葡萄球菌和铜绿假单胞菌的细胞壁上,成分可能为细菌细胞壁多糖.
목적 심조인수계세포촉발수체-1(TREM-1)적천연배체,위천명농독증적발병궤제제공이론의거.방법 분리인외주혈중성립세포화단핵세포진행원대배양.재량충분리배양적세포중분별가입열실활적금황색포도구균、동록가단포균、결핵분지간균혹고삼유도금황색포도구균L형、동록가단포균L형처리24 h.재량충분리배양적세포중분별가입초성제취적금황색포도구균세포벽、동록가단포균세포벽혹결핵분지간균세포벽처리24 h.재량충분리배양적세포중분별가입각세포벽삼대조분(세포벽다당、세포벽지류、세포벽단백)처리24 h.용형광정량취합매련반응(PCR)검측각처리조세포TREM-1 mRNA수평,용매련면역흡부법(ELISA)검측각처리조세포배양상청액중종류배사인자-α(TNF-α)화백세포개소-1β(IL-1β)농도.결과 금황색포도구균화동록가단포균적균체、세포벽화세포벽다당처리후중성립세포화단핵세포TREM-1mRNA수평이급세포배양상청액중TNF-α화IL-1β농도균명현승고.여공백대조조상비,금황색포도구균세포벽다당처리후중성립세포TREM-1 mRNA수평증가료(3.86±0.20)배,단핵세포TREM-1 mRNA수평증가료(5.15±0.56)배(균P＜0.05);동록가단포균세포벽다당처리후중성립세포TREM-1 mRNA수평증가료(4.03±0.15)배,단핵세포TREM-1 mRNA수평증가료(7.22±0.73)배(균P＜0.05).가입능경쟁성결합TREM-1배체적LP17가이삭약차효응;이결핵분지간균적균체、세포벽화세포벽조분균무차효응.결론 인TREM-1천연배체위우금황색포도구균화동록가단포균적세포벽상,성분가능위세균세포벽다당.
Objective To look for the natural ligand(s) of human triggering receptor expressed on myeloid cell-1 (TREM-1), in order to provide the theoretical basis for elucidation of the pathogenesis of sepsis.Methods Neutrophils and monocytes isolated from human peripheral blood were treated with heat-inactivated Staphylococcus aureus , Pseudomonas aeruginosa , Mycobacterium tuberculosis, Staphylococcus aureus L-form or Pseudomonas aeruginosa L-form respectively for 24 hours.The cell wall was extracted from Staphylococcus aureus, Pseudomonas aeruginosa and Mycobacterium tuberculosis by ultrasound.Neutrophils and monocytes were isolated and treated with the cell wall respectively for 24 hours.Neutrophils and monocytes were isolated and treated with three main components from bacterial cell wall (polysaccharides,lipids and proteins) respectively for 24 hours.The level of TREM-1 mRNA was measured with fluorescent quantitative polymerase chain reaction (PCR), and the concentrations of tumor necrosis factor-α (TNF-α)and interleukin-1β (IL-1β) were measured with enzyme-linked immunosorbent assay (ELISA).Results The TREM-1 mRNA level and the concentrations of TNF-α and IL-1β in cell supernatant of neutrophils and monocytes were upgraded when treated with cell, cell wall and cell wall polysaccharides of Staphylococcus aureus and Pseudomonas aeruginosa.Compared with the blank control group, the TREM-1 mRNA level of neutrophils and monocytes was upgraded to (3.86± 0.20)-fold and (5.15±0.56)-fold respectively when treated with cell wall polysaccharides of Staphylococcus aureus (both P＜0.05);the TREM-1 mRNA level of neutrophils and monocytes was upgraded to (4.03±0.15)-fold and (7.22±0.73)-fold respectively when treated with cell wall polysaccharides of Pseudomonas aeruginosa (both P＜0.05).The effect could be attenuated by the addition of LP17 which could bind TREM-1 ligand.This attenuating effect was not found when the cells were treated with cell, cell wall or cell wall polysaccharides of Mycobacterium tuberculosis.Conclusion The study provides the evidence that TREM-1 natural ligand (s) is present on cell wall of bacteria including Staphylococcus aureus and Pseudomonas aeruginosa, and it might be polysaccharides.