摘要
目的探讨吞噬经体外光化学法(PUVA)处理的同种异基因淋巴细胞的未成熟树突细胞(imDC)对脂多糖(LPS)介导的树突细胞(DC)激活的抑制作用。方法分离C57BL/6小鼠骨髓细胞,经小鼠重组白细胞介素4(rmIL-4)和重组粒-巨噬细胞集落刺激因子(rmGM-CSF)共同诱导培养制备骨髓来源的C57BL/6小鼠imDC。分离BALB/c小鼠脾淋巴细胞(SP),经PUVA处理制备成PUVA-SP。在体外将BALB/c小鼠的PUVA-SP与C57BL/6小鼠骨髓来源的imDC共同培养,获得PUVA-SP DCs。在上述imDC和PUVA-SP DCs中分别加入10ng/ml LPS刺激24h,获得LPS DCs。采用ELISA法检测上清中IL-10和IL-12的浓度,流式细胞仪检测细胞表面MHC-Ⅱ、CD40、CD86和CD80的表达水平。结果 LPS刺激后,PUVA-SP DCs表面分子CD80、CD86、CD40的表达分别为21.26%、38.50%和39.78%,远远低于imDC经LPS刺激后的表达(50.58%、66.29%、71.20%,P<0.01)。LPS刺激后PUVA-SP DCs和imDCs MHC-Ⅱ的表达分别为67.29%和68.64%,差异无统计学意义(P>0.05)。LPS刺激后,PUVA-SP DCs和imDCs分泌IL-10的水平分别为435.6±13.9pg/ml和132.6±2.8pg/ml,与刺激前相比均增高,但前者增高的水平要显著高于后者(P<0.01)。LPS刺激后,imDCs分泌高水平的IL-12,其p70和p40的表达量分别为192.1±5.9和999.8±26.9pg/ml,而PUVA-SP DCs分泌IL-12的水平分别为p7018.56±1.3pg/ml,p40 15.22±1.2pg/ml,显著低于imDCs,差异有统计学意义(P<0.01)。结论 PUVA-SP DCs虽然表达不成熟表型,但其功能不同于imDC,对LPS刺激具有抑制作用,适用于诱导体内免疫耐受。
Objective To investigate the inhibitory effect of immature dendritic cells（iDCs） on LPS-mediated maturation of iDCs phagocytizing allogeneic spleen lymphocytes after being treated by psoralen plus ultraviolet A（PUVA）.Methods Bone marrow-derived DCs were obtained from bone marrow cells of C57BL/6 mice by co-cultivation with recombinant mouse IL-4 and GM-CSF.Spleen lymphocytes（SLP） of BALB/c mice were isolated and transformed to PUVA-SLP by treatment with 8-methoxy PUVA irradiation.The bone marrow-derived iDCs of C57BL/6 were co-cultured with PUVA-SLP of BALB/c mice to obtain PUVASLP DCs.After incubation,iDCs and PUVA-SP DCs were induced to maturation by LPS（10ng/ml,24h）,and then they were analyzed by low cytometry.At the same time,the concentrations of the immunoreactive proteins IL-12p70,IL-12p40 and IL-10 in cell supernatants were determined by ELISA kits according to the manufacturer’s recommendations.Results PUVA-SLP DCs and iDCs were compared in terms of LPS responsiveness.The phenotype of iDCs（CD40,CD80,and CD86） was 50.58%,66.29%,71.20%,respectively,showed more rapid changes from immature to mature state in response to LPS stimulation compared with PUVA-SP DCs,the phenotype of which was 21.26%,38.50% and 39.78%,respectively（P＆lt;0.01）.After LPS stimulation,no difference was found in the expression of MHC-Ⅱbetween PUVA-SLP DCs and iDCs（67.29% and 68.64%,P＆gt;0.05）.PUVA-SP DCs secreted high levels of IL-10（435.6±13.9）,but low levels of IL-12（p70 18.56±1.3,p40 15.22±1.2）,as compared with those of iDCs（132.6±2.8,p70 192.1±5.9,p40 999.8±26.9,P＆lt;0.01） after LPS stimulation.Conclusions Although PUVA-SLP DCs do not express as immature phenotype,they can be readily induced to differentiate into mature DCs in the presence of antigen or LPS.It may be suitable to use iDCs clinically in autoimmune diseases and transplantation.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2013年第8期625-628,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金青年基金(81102242)
北京市科技专项2011阶梯计划(Z111102055311086)
中国博士后基金(200902680
20080441308)~~
关键词
树突细胞
免疫耐受
脂多糖类
dendritic cells
immune tolerance
lipopolysaccharides
