CAJ | 학술논문

目的：评估小鼠不同组织中B10细胞的比例，分离B10细胞并鉴定其生物学功能，探讨其对效应性T细胞（ CD4＋CD25-T）及调节性T细胞（ Treg）的免疫调节机制。方法采用流式细胞仪检测小鼠不同组织来源B10细胞的表达以及脂多糖（ lipopolysaccharide ，LPS）对B10激活的影响；采用流式细胞分选术（ FACS）和免疫磁珠分选术（ MACS）分选B10细胞、CD4＋CD25-T 细胞和Treg；利用混合淋巴细胞培养实验观察B10对CFSE标记的CD4＋CD25-T细胞和CD4＋CD25＋T细胞增殖的调节作用及机制。结果（1）脾脏CD19＋CD5＋CD1dhigh B细胞表达为（3．95±0．79）％，与外周血、肠系膜淋巴结和外周淋巴结相比较表达最高，组间差异有统计学意义（P＜0．05）；脾脏CD19＋IL-10＋B细胞表达为（2．02±0．16）％，与外周血、肠系膜淋巴结和外周淋巴结相比较表达最高（P＜0．05），且IL-10主要由CD1dhighCD5＋B细胞亚群分泌（P＜0．01）；（2） LPS联合乙酸佛波醇（PMA）、莫能菌素（monen-sin）和离子霉素（ionomycin）能活化B10细胞分泌IL-10。延长LPS的作用时间（48 h）能促进CD19＋CD5＋CD1dhigh B细胞高表达及IL-10分泌增加（P＜0．01）；（3） CD19＋CD5＋CD1dhigh B细胞在体外可抑制CD4＋CD25-T细胞增殖（P＜0．01）、促进CD4＋T细胞分泌IL-10（P＜0．01），同时可促进Treg增殖（P＜0．01）。结论 B10细胞在小鼠脾脏高表达，通过Toll样受体（TLR）信号通路被激活。活化的B10细胞具有免疫抑制性，可抑制CD4＋CD25-T细胞增殖，促进Treg增殖，其可能免疫调节机制是IL-10的释放增加。因此B10细胞有望为治疗多种自身免疫性疾病提供一种全新的治疗方法。
목적：평고소서불동조직중B10세포적비례，분리B10세포병감정기생물학공능，탐토기대효응성T세포（ CD4＋CD25-T）급조절성T세포（ Treg）적면역조절궤제。방법채용류식세포의검측소서불동조직래원B10세포적표체이급지다당（ lipopolysaccharide ，LPS）대B10격활적영향；채용류식세포분선술（ FACS）화면역자주분선술（ MACS）분선B10세포、CD4＋CD25-T 세포화Treg；이용혼합림파세포배양실험관찰B10대CFSE표기적CD4＋CD25-T세포화CD4＋CD25＋T세포증식적조절작용급궤제。결과（1）비장CD19＋CD5＋CD1dhigh B세포표체위（3．95±0．79）％，여외주혈、장계막림파결화외주림파결상비교표체최고，조간차이유통계학의의（P＜0．05）；비장CD19＋IL-10＋B세포표체위（2．02±0．16）％，여외주혈、장계막림파결화외주림파결상비교표체최고（P＜0．05），차IL-10주요유CD1dhighCD5＋B세포아군분비（P＜0．01）；（2） LPS연합을산불파순（PMA）、막능균소（monen-sin）화리자매소（ionomycin）능활화B10세포분비IL-10。연장LPS적작용시간（48 h）능촉진CD19＋CD5＋CD1dhigh B세포고표체급IL-10분비증가（P＜0．01）；（3） CD19＋CD5＋CD1dhigh B세포재체외가억제CD4＋CD25-T세포증식（P＜0．01）、촉진CD4＋T세포분비IL-10（P＜0．01），동시가촉진Treg증식（P＜0．01）。결론 B10세포재소서비장고표체，통과Toll양수체（TLR）신호통로피격활。활화적B10세포구유면역억제성，가억제CD4＋CD25-T세포증식，촉진Treg증식，기가능면역조절궤제시IL-10적석방증가。인차B10세포유망위치료다충자신면역성질병제공일충전신적치료방법。
Objective To investigate the phenotypes and percentages of B 10 cells in different tis-sues from wild-type mice and to identify their biological functions .Methods The percentages of B10 cells derived from different tissues of mice and their responses to lipopolysaccharide ( LPS) stimulation were ana-lyzed by flow cytometry .Magnetic-activated cell sorting ( MACS ) and fluorescence-activated cell sorting (FACS) were used to purify B10 cells, CD4+CD25-T cells and Treg cells.CD4+CD25-T cells and Treg cells labeled by CFSE were co-cultured with or without B10 cells, and then their proliferation were evaluated after 72 h.Results (1) A subset of CD19+CD5+CD1dhigh regulatory B cells was identified in spleen , pe-ripheral blood and lymph nodes from wild-type mice , of which the highest frequency was detected in spleen (3.95%±0.79%, P<0.05).The isolated B cells from different tissues were stimulated by LPS , PMA, ionomycin and monensin (L+PIM) in vitro to express IL-10.Among them, splenic CD19+IL-10+B cells showed the highest expression of IL-10 (P<0.05).(2) Prolonged LPS stimulation (48 h) to CD5+CD1dhigh B cells enhanced the expressions of IL-10 (P<0.01).(3) CD19+CD5+CD1dhigh B cells inhibited the prolif-eration of CD4+CD25-T cells in vitro in a dose-dependent manner (P<0.01), but increased the secretion of IL-10 by CD4+T cells (P<0.01) and the proliferation of Treg cells in vitro (P<0.01).Conclusion Com-pared with other tissues , the percentage of B10 cell subset in spleen is the highest in wild-type mouse , and B10 cells subset can be activated through Toll-like receptor ( TLR ) signaling pathway .The responses of CD4+CD25-T cells and Treg cells in co-culture with B10 cells are regulated by B 10 cell subset through an increased IL-10 production .B10 cells might be a useful cell population for the treatment of inflammatory au-toimmune diseases.