银耳多糖改善脓毒症小鼠调节性T细胞的免疫活性

摘要

Objective:To determine the effects of TPS on peripheral blood Tregs in sepsis mouse induced by burn plus P.aeruginosa infection.Methods: The experimental mice were separated into five groups randomly ,including sham burn group ,burn plus P.aeruginosa infection group ,burn plus P.aeruginosa infection with TPS (50,100,200 mg/kg) treatment group.Peripheral blood Tregs were isolated with Magnetic Microbeads and cultured in vitro from the day after burn (PBD0) to 4 days after burn(PBD4).IL-10, IFN-γ,IL-4 levels in Tregs culture supernatants were determined by sandwich enzyme-linked immunosorbent assays ( ELISA ) . Purification of CD4+CD25high Tregs and CD4+T cells in C57BL/6 mice were administrated by magnetic beads sorting .Tregs and CD4+T cells were cultured in vitro after joining TPS to without TPS cells as a control .The phenotypes of Tregs were analyzed by flow cytometry , and cytokines were measured by ELISA .Results:Vis-a-vis the results of the untreated group ,TPS could markedly decrease IL-4 and IL-10 secretion level and significantly increase the secretion of IFN-γ,and the secretion of IL-10 level and concentration of TPS dose effect.Vis-a-vis the results of the untreated group ,in vitro experiment ,without stimulation of TPS ,CD4+T cell proliferation and IFN-γwere significantly reduced ( P<0.05 ) and IL-4 levels increased significantly;CD4+T cell proliferation and IFN-γwere significantly increased and IL-4 levels were significantly reduced in the group of TPS with antibody-1;there was no significant difference in CD 4+T cell proliferation and the levels of IFN-γand IL-4 in the group of TPS with antibody-2.Conclusion:TPS could inhibit the abnormal ac-tivities of CD4+CD25highTregs in burn with P.aeruginosa infection mice,at least in part via inhibiting IL-10 secretion,and trigger a shift of Th2 to Th1 with activation of CD4+T cells in burn with P.aeruginosa infection mice.%目的：探讨银耳多糖（ TPS）对脓毒症小鼠外周血调节性T细胞免疫调节的作用。方法：雄性BALB／c小鼠（20±1）g，采用烧伤后腹腔注射铜绿假单胞菌制作脓毒症并予以TPS干预，随机分成5组，分别为正常对照组（未做任何处理），未刺激组（烧伤后铜绿假单胞菌感染未经TPS处理组）， TPS低剂量组（烧伤后铜绿假单胞菌感染＋50 mg／kg治疗组）， TPS中剂量组（烧伤后铜绿假单胞菌感染＋100 mg／kg治疗组），TPS高剂量组（烧伤后铜绿假单胞菌感染＋200 mg／kg治疗组），从各组的外周血中分离调节性T细胞，并体外培养。自烧伤即刻（ PBD0）起，至烧伤后4 d每天监测培养上清液中IL－10、IFN－γ、IL－4的水平。磁珠孵育和磁性分离小鼠外周血的调节性T细胞（CD4＋CD25high Tregs）和CD4＋T细胞，分别加入TPS后进行培养，以不加TPS的细胞作为对照，流式细胞仪分析CD4＋CD25 high Tregs的表型，ELISA法检测细胞因子的表达。结果：与对照组相比，TPS能显著降低未刺激组小鼠的IL－10和IL－4的分泌，并显著增加IFN－γ的分泌，并且IL－10的分泌水平与TPS浓度呈剂量效应关系。体外培养试验中，与正常对照组相比，未刺激组的CD4＋T细胞增殖和IFN－γ水平显著降低（P＜0．05），IL－4水平显著升高（P＜0．05）；与未刺激组相比，TPS刺激组的CD4＋T细胞增殖和IFN－γ水平显著升高（P＜0．05），IL－4水平显著降低（P＜0．05）；与未刺激组相比，TPS刺激＋抗体1组CD4＋T细胞增殖和IFN－γ水平显著升高（P＜0．05），IL－4水平显著降低（P＜0．05）；与未刺激组相比，TPS刺激＋抗体2组的CD4＋T细胞增殖和IFN－γ、IL－4水平无显著性差异。结论：TPS可以通过降低IL－10的分泌抑制CD4＋CD25 high Tregs对CD4＋T淋巴细胞增殖和极化的影响，并诱导CD4＋T淋巴细胞向Th1分型，从而使机体免疫活性增强。