吞噬PUVA处理的同种异基因细胞的树突细胞对抗原特异性CD4^+ CD25^+ Foxp3^+调节T细胞的体外诱导作用  

Induction effects of dendritic cells loaded with PUVA-treated allogeneic cells on alloantigen-specific CD4^+ CD25^+ Foxp3^+ T regulatory cells in vitro

作　　者：郑德华[1] 陆宏[2] 石炳毅[1] 杜国盛[1] 邹一平[1] 魏玉香[1] 窦立萍[3] 史迎昌[4] 黎力[4] 洪宝发[5] 

机构地区：[1]解放军309医院全军器官移植中心,北京100091 [2]解放军309医院心内科,北京100091 [3]解放军总医院血液病科,北京100853 [4]解放军309医院动物实验室,北京100091 [5]解放军总医院泌尿外科,北京100853

出　　处：《解放军医学杂志》2010年第5期543-546,共4页Medical Journal of Chinese People's Liberation Army

基　　金：全军"十一五"科技攻关项目(06G115);北京市自然科学基金(7072078;7102147);解放军总医院科技创新苗圃基金(09KMM32)

摘　　要：目的探讨吞噬体外光化学法(PUVA)处理的同种异基因淋巴细胞的树突细胞(DC)对抗原特异性CD4+ CD25 +Foxp3+调节性T细胞的体外诱导作用。方法分离LEW大鼠骨髓细胞,经大鼠重组IL-4和GM-CSF共同诱导培养制备骨髓来源的LEW大鼠DC。分离DA大鼠脾淋巴细胞(SP),制备经PUVA处理的DA大鼠脾淋巴细胞(PUVA-SP)并用流式细胞仪检测其凋亡率。在体外将DA大鼠的PUVA-SP或SP与LEW大鼠骨髓来源的DC共同培养,得到PUVA-SPDC和SPDC,以Luminex液相芯片法检测PUVA-SPDC和SPDC培养上清中IL-10、IL-12的含量。以CFSE标记PUVA-SP,流式细胞仪检测LEW大鼠DC对DA大鼠PUVA-SP的摄取情况。将LEW大鼠CD4+25-T细胞与PUVA-SPDC或SPDC混合培养5d,流式细胞仪检测诱导形成的CD4+ CD25+ Foxp3+ T细胞,同时分离培养体系中的CD4+ CD25+ T细胞,通过混合淋巴细胞反应(MLR)检测PUVA-SPDC诱生的CD4+ CD25+ T细胞对LEW大鼠CD4+ CD25- T细胞(效应性T细胞)体外增殖的抑制作用。结果DA大鼠脾淋巴细胞经PUVA处理后细胞凋亡率为81.93%。LEW大鼠DC可有效摄取PUVA-SP,吞噬率达70.72%。SPDC培养上清中IL-12和IL-10浓度分别为700±18、39±3pg/ml;与SPDC相比,PUVA-SPDC培养上清中的IL-12水平(148±9pg/ml)明显降低(P<0.01),而IL-10水平(75±4pg/ml)则明显增加(P<0.01)。与PUVA-SPDC混合培养后,LEW大鼠CD4+T细胞中CD4+ CD25+ T细胞的比例为15.4%,其中Foxp3阳性率为44.1%;而与SPDC混合培养后LEW大鼠CD4+T细胞中CD4+ CD25+ T细胞的比例只有6.5%,其中Foxp3阳性率仅为8.2%。PUVA-SPDC诱生的CD4+ CD25+ T细胞对体外增殖反应有明显的抗原特异性抑制作用。结论PUVA-SPDC在体外能够有效诱生抗原特异性CD4+ CD25+ Foxp3+调节性T细胞。Objective To investigate the induction effects in vitro of dendritic cells （DC） loaded with psoralen plus ultraviolet A （PUVA） treated allogeneic spleen lymphocytes on alloantigen-specific CD4＋ CD25＋ Foxp3＋ T regulatory cells （Treg）. Methods Bone marrow-derived DC of LEW rat was obtained from bone marrow cells by co-culturing with recombinant rat IL-4 and GM-CSF. Spleen lymphocytes （SP） of DA rat were isolated and prepared to PUVA-SP by treating with 8-methoxypsoralen plus ultraviolet A irradiation, and the apoptosis rates were determined by flow cytometry. The bone marrow-derived DC of LEW rat was co-cultured with PUVA-SP or SP of DA rat to obtain PUVA-SP DC or SP DC, and the contents of IL-10 and IL-12 in the supernatant were determined by Luminex method. The PUVA-SP was labeled with CFSE, and the PUVA-SP uptake of DC was detected by flow cytometry. The CD4＋CD25-T cells derived from normal LEW rat were co-cultured with PUVA-SP DC or SP DC for 5 days, then the frequency of CD4＋ CD25＋ Foxp3＋ T cells was determined by flow cytometry, while the CD4＋ CD25＋ T cells were isolated from the culture system to determine its suppressive effects on CD4＋CD25-T cells derived from normal LEW rat （effector T cells） by mixed lymphocyte reaction （MLR）. Results The apoptotic rate of DA rat spleen lymphocytes treatmented with PUVA was 81.93%. DC derived from LEW rat could uptake PUVA-SP efficiently in a phagocytosis rate of 70.72%. The concentration of IL-12 in supernatant of PUVA-SP DC （148±9pg/ml） was significantly lower than that of SP DC （700±18pg/ml, P0.01）, while the concentration of IL-10 in supernatant of PUVA-SP DC （75±4pg/ml） was higher than that of SP DC （39±3pg/ml, P0.01）. After co-culturing with PUVA-SP DC, the percentage of CD4＋CD25＋ T cells in CD4＋ T cells was 15.4%, and the Foxp3 positive rate of CD4＋CD25＋ T cells was 44.1%. Meanwhile, after co-culturing with SP DC, the percentage of CD4＋CD25＋ T cells in CD4＋ T cells was only 6.5%, and t

关 键 词：树突细胞 PUVA疗法 T淋巴细胞 调节性 转录因子FOXP3 

分 类 号：R392.4[医药卫生—免疫学]