树突状细胞吞噬经光化学处理的异基因细胞后对同基因T细胞的免疫调控  

Immune Regulation Effect of Rat Dendritic Cells Phagocytosing Photochemotherapy-treated Allogeneic Cells on Syngeneic T Cells

作　　者：郑德华[1] 魏玉香[1] 石炳毅[1] 邹一平[1] 杜国盛[1] 朱志东[1] 宋继勇[1] 史迎昌[2] 黎立[2] 

机构地区：[1]解放军总医院第二附属医院全军器官移植中心,北京100091 [2]解放军总医院第二附属医院动物实验室,北京100091

出　　处：《中国实验血液学杂志》2009年第6期1492-1496,共5页Journal of Experimental Hematology

基　　金：全军"十一五"计划科技攻关项目;编号06G115;北京市自然科学基金面上课题;编号7072078

摘　　要：本研究探讨树突状细胞(DC)吞噬经PUVA(8-methoxypsoralen plus UVA irradiation,8-甲氧基补骨脂素联合长波紫外线照射的体外光化学治疗)处理的同种异基因淋巴细胞后对于同基因T细胞的免疫调控作用。分离LEW大鼠骨髓细胞,经IL-4和GM-CSF共同诱导制备骨髓来源的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),检测PUVA-SP的凋亡率。在体外将PUVA-SP或SP与受者骨髓来源的未成熟DC共同培养,检测上述处理对受者DC表面分子CD40、CD86、MHC-Ⅱ的影响。以CFSE标记PUVA-SP,通过荧光显微镜观察LEW大鼠DC吞噬DA大鼠PUVA-SP的情况。通过混合淋巴细胞培养(MLR),检测吞噬DA大鼠PUVA-SP的LEW大鼠DC(PUVA-SPDC)对LEW大鼠T细胞的刺激作用,同时检测MLR培养上清中细胞因子IL-4、IL-10、IL-2、IFN-γ的浓度。结果表明:PUVA处理能够在24小时内诱导62.87%的DA大鼠脾淋巴细胞发生早期凋亡。LEW大鼠DC与DA大鼠PUVA-SP共培养后,可有效吞噬PUVA-SP。LEW大鼠DC与DA大鼠SP混合培养后,其表面分子MHC-Ⅱ、CD40以及CD86的表达阳性率分别为65.6%、45.6%和36.5%,明显高于未经处理的受者DC组27.7%、22.9%和13.0%(p<0.01);而DC与PUVA-SP混合培养后,其表面分子MHC-Ⅱ、CD40及CD86的表达仍保持较低的水平,分别为25.7%、21.0%和13.4%,与未经任何处理的对照组DC相当(p>0.05),而远远低于LPS刺激后的成熟DC(82.3%、71.7%和63.2%)(p<0.01)。DA大鼠SP共培养后的LEW大鼠DC可以刺激LEW大鼠T细胞显著增殖,刺激指数在加载DC数量为2×103、10×103、20×103组分别为1.7546、3.9798和5.0220,而吞噬DA大鼠PUVA-SP的LEW大鼠DC则诱导了T淋巴细胞低反应性,刺激指数分别为1.0120、1.1518和1.4093,二者差异有统计学意义(p<0.01)。此外,与SPDC相比,PUVA-SPDC明显抑制了T细胞IFN-γ的分泌(140.17±2.28vs37.67±1.76)(p<0.01),刺激了IL-10(33.11±1.82vs69.58±1.83)(p<0.01)、IL-4的分泌(11.11±1.07vs23.83±0.73)(p<The aim of this study was to investigate the immune regulatory effect of dendritic cells phagocytosing photochemotherapy-treated allogeneic spleen lymphocytes on syngeneic T cells. DA rat spleen lymphocytes were treated with 8-methoxypsoralen plus UVA irradiation （PUVA）. LEW rat bone marrow-derived DCs were co-cultured with PUVA-treated DA spleen lymphocytes （PUVA-SP）, and the surface markers （MHC-Ⅱ, CD86 and CD40 ） of treated DC were detected by flow cytometry. CFSE-labeled PUVA SP were incubated with LEW DCs and the phagocytosis of DCs on PUVA-SP was observed by using fluorescent microscope. The ability of DC phagocytosing allogeneic PUVA-SP （PUVA-SP DC） to stimulate the proliferation of LEW T cells was analyzed by mixed leukocyte reactions （MLR）. The production of IL-4, IL-10, IL-2, IFN-γ in MLR culture supernatant was determined by luminex method. The results indicated that the PUVA treatment effectively induced early apoptosis of DA rat spleen lymphocytes. After co-culture, DC efficiently phagocytosed allogeneic pLrVA-SP and still maintained an immature phenotype with low levels of MHC Ⅱ, CD40 and CD86. PUVA-SP DC induced LEW T cell hyporesponsiveness to DA rat antigen, and led to skewing of T cell cytokine expression toward Th2 （IL-10 and IL-4）. It is concluded that the PUVA-SP DC effectively down-regulate T cell response to alloantigen and induce Th2 immune deviation in vitro.

关 键 词：树突状细胞 PUVA 免疫调控 免疫偏移 

分 类 号：R392.12[医药卫生—免疫学] R392.11[医药卫生—基础医学]