阻断CD40-CD40L共刺激途径对T细胞表型及细胞因子分泌的作用研究  

The study of the role of blockade of CD40 ligand-CD40 costimulatory pathway on T lymphocytes typing and cytokines

作　　者：陈纯[1] 黄绍良[1] 段连宁[2] 李浩威[2] 温冠媚[2] 魏菁[3] 

机构地区：[1]中山大学附属第二医院儿科,广州510120 [2]中山大学医学院病理生理教研室,广州510120 [3]中山大学附属第二医院医学中心,广州510120

出　　处：《中国免疫学杂志》2003年第8期534-537,共4页Chinese Journal of Immunology

基　　金：美国中华医学会基金资助 (CMB) (960 3 60 )

摘　　要：目的 :探讨应用抗CD4 0L单克隆抗体阻断CD4 0 CD4 0L共刺激途径后对T细胞表型及其分泌的细胞因子的影响 ,为体外阻断该共刺激途径诱导T细胞对异体移植抗原的免疫耐受提供实验依据。方法 :供鼠 (C5 7BL 6 H 2b)脾T细胞作为反应细胞 ,受鼠 (BALB CH 2d)脾细胞作为刺激细胞 ,设单抗组 (加抗CD4 0L单抗 )和对照组 (不加单抗 ) ,初次混合淋巴细胞培养(MLR) 7天 ,在不同时间点采用3H TdR掺入法检测细胞增殖率 ,以ELISA法测定培养上清液中IFN γ、IL 2、IL 4、IL 10等的水平 ,第 5天采用流式细胞仪检测CD4 + T和CD8+ T细胞上CD2 5、CD6 9、CD4 0L和CD4 5RA的表达。再次MLR 5天 ,第 1、3、5天采用3H TdR掺入法测定细胞的增殖情况和ELISA法测定培养上清液中的上述细胞因子的水平。结果 :初次和再次MLR结果均显示 ,单抗组细胞增殖反应率明显低于对照组。初次MLR单抗组中CD4 + T和CD8+ T细胞比例明显低于对照组 (P <0 0 5 ) ;单抗组中CD4 + CD2 5 + T、CD4 + CD6 9+ T、CD8+ CD2 5 + T、CD4 + CD4 0L+ T和CD8+ CD6 9+ T细胞比例明显低于对照组 (P <0 0 5 ) ,而CD8+ CD4 0L+ T和CD4 + CD4 5RA +T细胞的比例与对照组相比无明显差异 (P >0 0 5 )。初次MLR中单抗组和对照组培养上清中IL 4和IL 10几乎无法测出 。Objective:To investigate the role of blockade of CD40 ligand-CD40 costimulatory pathway by anti-CD40L mAb on T lymphocytes typing and cytokines, and provide the experimental clues of inducing donor-specific T cell anergy.Methods:To monitor primary MLR, C57BL/6 H-2b spleen T cells were isolated as responder cells, and BALB/C H-2d spleen cell as stimulator cells. Anti-CD40L mAb was added to primary MLR cultures(termed anti-CD40L mAb group), and in control group(no anti-CD40L mAb). Incubated for 7 days, the cells responsiveness rates were detected by 3 H-TdR methods at the indicated time points, and supernatants were assayed for IFN-γ,IL-2,IL-4,IL-10 cytokines levels by commercial ELISA kits. Day 5 MLR-cultured cells were assessed for the expressions of CD4, CD8, CD25, CD69, CD40L and CD45RA with flow cytometry(FCM).Cells proliferation for 5 days and cytokines in secondary MLR were assayed according above-mentioned method.Results:Blockade of the CD40-CD40L pathway by anti-CD40L mAb can induce the hyporesponsiveness to alloantigen in primary and secondary MLR culture. In primary MLR culture, the expressions of CD4 +T and CD8 +T cells and CD4 +CD25 +T, CD4 +CD69 +T, CD4 +CD40L +T, CD8 +CD25 +T and CD8 +CD69 +T cells in anti-CD40L mAb group were lower than that in control group ( P <0.05). The expression of CD8 +CD40L +T were similar in two groups( P >0.05).The expression of CD4 +CD45RA + T in anti-CD40L mAb-cultured group was higher than that in control group( P <0.05). The levels of IL-4 and IL-10 in supernatant of the primary MLR culture were undetectable in two groups. The levels of IFN-γ and IL-2 in supernatants of the anti-CD40L mAb-cultured primary MLR were significantly lower than that of the control group ( P <0.01). The levels of IFN-γ 、 IL-2 and IL-4、IL-10 in supernatants of the anti-CD40L mAb-cultured secondary MLR were lower than that of the control group ( P <0.05).Conclusion:In a MLR system,anti-CD40L mAb cocultured with donor spleen T cells could act on the CD4 + an

关 键 词：CD40-CD40L T细胞表型 细胞因子 分泌作用 免疫耐受 

分 类 号：R457.2[医药卫生—治疗学]