CAJ | 학술논문

目的：探讨调节性B细胞（ Breg）在川崎病（ KD）免疫发病机制中的作用。方法急性期KD患儿28例，正常同年龄对照组28例，KD患儿分别于静脉丙种球蛋白（ IVIG）治疗前后直接取血备检。采用流式细胞术分别检测外周血CD19＋CD24 hi CD38 hi Breg、CD4＋CD25＋Foxp3＋调节性T细胞（ Treg）比例及Breg细胞表面共刺激分子CD80、CD86、PD-L1的表达；酶联免疫吸附实验检测培养上清中IL-10蛋白浓度；实时荧光定量PCR （real-time PCR）检测外周血B细胞中IL-10，CD4＋T细胞中Foxp3、CTLA-4和GITR mRNA的表达。结果（1）急性期KD患儿外周血CD19＋CD24hiCD38hi Breg细胞比例及B细胞IL-10表达均明显低于同年龄对照组（P＜0．05），经IVIG治疗后显著上升（P＜0．05）；经体外LPS刺激培养48 h后，急性期KD患儿B细胞培养上清中IL-10浓度仍明显低于同年龄正常对照组[（31．06±8．26） pg/ml vs （46．32±13．91） pg/ml， P＜0．05]；（2）急性期KD患儿CD19＋CD24hiCD38hi Breg细胞表面共刺激分子CD80、CD86、PD-L1表达明显下调（P＜0．05），经IVIG治疗后，CD80和CD86表达虽有所增加，但仍低于同年龄对照组（P＜0．05），PD-L1表达无明显改变（P＞0．05）；（3）急性期KD患儿外周血CD4＋CD25＋Treg细胞比例及相关分子Foxp3、CTLA-4和GITR基因转录水平明显低于正常对照组（P＜0．05），其中CD4＋CD25＋Treg细胞比例与CD19＋CD24hiCD38hi Breg细胞比例呈正相关关系（r＝0．62，P＜0．05），经IVIG治疗后显著上升（P＜0．05）。结论 Breg细胞数量及功能异常可能是导致急性期KD患儿免疫功能紊乱的重要原因之一。
목적：탐토조절성B세포（ Breg）재천기병（ KD）면역발병궤제중적작용。방법급성기KD환인28례，정상동년령대조조28례，KD환인분별우정맥병충구단백（ IVIG）치료전후직접취혈비검。채용류식세포술분별검측외주혈CD19＋CD24 hi CD38 hi Breg、CD4＋CD25＋Foxp3＋조절성T세포（ Treg）비례급Breg세포표면공자격분자CD80、CD86、PD-L1적표체；매련면역흡부실험검측배양상청중IL-10단백농도；실시형광정량PCR （real-time PCR）검측외주혈B세포중IL-10，CD4＋T세포중Foxp3、CTLA-4화GITR mRNA적표체。결과（1）급성기KD환인외주혈CD19＋CD24hiCD38hi Breg세포비례급B세포IL-10표체균명현저우동년령대조조（P＜0．05），경IVIG치료후현저상승（P＜0．05）；경체외LPS자격배양48 h후，급성기KD환인B세포배양상청중IL-10농도잉명현저우동년령정상대조조[（31．06±8．26） pg/ml vs （46．32±13．91） pg/ml， P＜0．05]；（2）급성기KD환인CD19＋CD24hiCD38hi Breg세포표면공자격분자CD80、CD86、PD-L1표체명현하조（P＜0．05），경IVIG치료후，CD80화CD86표체수유소증가，단잉저우동년령대조조（P＜0．05），PD-L1표체무명현개변（P＞0．05）；（3）급성기KD환인외주혈CD4＋CD25＋Treg세포비례급상관분자Foxp3、CTLA-4화GITR기인전록수평명현저우정상대조조（P＜0．05），기중CD4＋CD25＋Treg세포비례여CD19＋CD24hiCD38hi Breg세포비례정정상관관계（r＝0．62，P＜0．05），경IVIG치료후현저상승（P＜0．05）。결론 Breg세포수량급공능이상가능시도치급성기KD환인면역공능문란적중요원인지일。
Objective To investigate the role of regulatory B cells ( Breg ) in the immunological pathogenesis of Kawasaki disease ( KD) .Methods Twenty-eight children with acute KD and twenty-eight age-matched healthy subjects were enrolled in this study .Blood samples were collected before and after the treatment of intravenous immunoglobulin (IVIG).The proportions of CD19+CD24hiCD38hi Breg and CD4+CD25+Foxp3+regulatory T cells (Treg), and the expressions of co-stimulatory molecules (CD80, CD86 and PD-L1) were analyzed by flow cytometry .The concentration of IL-10 protein was measured by enzyme-linked immunosorbent assay .Real-time PCR was performed to evaluate the expressions of Foxp 3, CTLA4 and GITR in CD4+T cells and IL-10 in CD19+B cells at mRNA level.Results (1) The proportions of CD19+CD24hi CD38hi Breg in peripheral blood and the expressions of IL-10 at mRNA level in CD19+B cells from patients with KD were lower than those from healthy controls (P<0.05), but significantly increased after treated with IVIG (P<0.05).Upon stimulation of lipopolysaccharide for 48 hours, the concentrations of IL-10 protein in culture supernatant of B cells from patients with KD were still lower than those from healthy controls [(31.06±8.26) pg/ml vs.(46.32±13.91) pg/ml, P<0.05].(2) Compared with healthy controls , the expressions of CD80, CD86 and PD-L1 were remarkably down-regulated in patients with acute KD ( P<0.05).With the treatment of IVIG, the expressions of CD80 and CD86 were significantly up-regulated though lower than those of the control group (P<0.05).There was no significant difference in the expression of PD-L1 before and after treatment (P>0.05).(3) The proportions of CD4+CD25+Foxp3+Treg and the expressions of Foxp3, CTLA-4 and GITR at mRNA level were significantly decreased in KD group compared with those in control group (P<0.05), but were increased to some extent after IVIG treatment (P<0.05). In addition, a positive correlation between the proportion of CD 19+CD24hiCD38hi Breg and the proportion of CD4+CD25+Foxp3+Treg was found in patient with acute KD (r=0.62, P<0.05).Conclusion Breg cell deficiency and its impaired function might be one of the important factors causing immune dysfunction in pa -tients with acute KD .