幼年类风湿关节炎血清对小鼠树突状细胞LTB4-BLT2通路的影响与探讨

目的 探讨幼年类风湿关节炎(JRA)血清对成熟小鼠树突状细胞(DCs)白三烯B4(LTB4)及其受体2(BLT2)信号通路(LTB4-BLT2)的影响.方法 分离正常小鼠骨髓细胞,体外用细胞因子重组小鼠白细胞介素4[(rmIL-4),重组小鼠粒细胞巨噬细胞集落因子(rmGM-CSF)]诱导、分化培养成幼稚DCs,细菌脂多糖(LPS)刺激其成熟.光学显微镜观察其形态,流式细胞术对其进行鉴定;酶联免疫吸附试验(ELISA)测定树突状细胞分化成熟过程中培养上清、正常血清组、JRA活动期血清组和BLT2阻断剂(LY255283)作用组中LTB4含量;免疫细胞化学方法和免疫荧光抗体法检测成熟DCs BLT2的定位表达、反转录-聚合酶链反应(RT-PCR)检测其mRNA表达;流式细胞术检测正常血清组、JRA活动期血清组和BLT2阻断剂作用于成熟DCs 18 h后BLT2的表达.结果 成熟DCs存在LTB4-BLT2信号通路表达;DCs不仅存在BLT2的基因mRNA表达,而且存在蛋白表达,蛋白表达于DCs细胞膜和胞质,以胞质细胞器表达最强;正常血清组、JRA活动期血清组、BLT2阻断剂组作用于成熟DC 18 h前后LTB4含量分别为(17±3)pg/ml、(82±20)pg/ml、(82±20)pg/ml和(24±6)pg/ml、(115±20)pg/ml、(91±11)pg/ml,JRA活动期血清组与正常血清组比较LTB4含量明显增高(P＜0.01),BLT2阻断剂组与JRA活动期血清组比较LTB4含量下降(P<0.05).同时,正常血清组、JRA活动期血清组和BLT2阻断剂组DCs BLT2表达分别为27.7±2.9、46.3±8.7和30.3±5.5,JRA活动期血清组与正常血清组比较,BLT2表达明显增强(P<0.05);当JRA活动期血清中加入BLT2阻断剂后,与JRA活动期血清组比较BLT2表达则下降(P<0.05).结论 DCs可能通过其BLT2与自分泌和(或)外在的:LTB4建立LTB4-BLT2炎症信号通路联系,该信号通路增强可能参与了JRA的发生及活动过程.

The effect of juvenile rheumatoid arthritis serum on the LTB4-BLT2 signal pathway of mice dendritic cells

Objective To investigate the effect of iuvenile rheumatoid arthritis(JRA) serum (leukotriene B4,LTB4) LTB4-BLT2 on mice DCs.Methods Bone marrow(BM)-derived DCs from healthy mouse were purified.and induced by cytokine IL-4 and GM-CSF to immature DCs and then differentiated to mature DCs under the stimulation of LPS in vitro.DCs were evaluated by light microscope and flow cvtometry.The concentrations of LTB4 in DCs supernatant,normal serum,active JRA,and that of the co-cuItured with BLT2 antagonist LY255283 were detected by ELISA.The expression of BLT2 protein and mRNA in DCs was examined by immunocytochemistry,immunofluorescence and RT-PCR.Meanwhile,the expression of BLT2 in DCs after 18 h co-cultured with normal serum,in serum of active JRA and that of BLT2 antagonist (LY255283)group was assayed by flow cytometry respectively.Results LTB4-BLT2 was expressed by DCs.Not only BLT2 mRNA but also its protein was expressed in DCs.The concentration of LTB4 Was(17±3)pg/ml,(82±20)pg/ml,(82±20)pg/ml and(24±6)pg/ml,(115±20)pg/ml,(91±11)pg/ml in normal serum group,active JRA group and LY255283 group before and after 1 8 h,respectively.The expression Was higher in serum of active JRA group than that of normal sertlm group(P<0.01)and there was a tendency to be higher when compared with LY255283 group(P<0.05).The DC BLT2 expression was 27.7±2.9,46.3±8.7 and 30.3±5.5 in normal serum group,serum of active JRA group and LY255283 grbup after 1 8h respectively.The expression was stronger in active JRA group than other groups(P<0.05).Conclusion DC can develop a LTB4-BLT2 signal pathway by BLT2 with autocrine and/or extrinsic LTB4.The overexpression of this pathway may be involved in the initiating and activation of JRA.