甲氧基聚乙二醇衍生物与抗OX40L单抗双诱导移植物免疫耐受  

Immune tolerance of transplants induced by the combination of methoxy polyethylene glycol and anti-OX40L monoclonal antibody

作　　者：冯洒然[1] 黄一虹[1] 杜冰[1] 潘秀英[1] 徐开林[1] 

机构地区：[1]徐州医学院附属医院血液科,江苏省徐州市221002

出　　处：《中国组织工程研究与临床康复》2008年第29期5663-5667,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江苏省高校"青蓝工程"优秀青年骨干教师资助项目(sjs200512)~~

摘　　要：背景:甲氧基聚乙二醇可在淋巴细胞表面形成空间位阻而遮蔽T细胞表面抗原;OX40及其配体OX40L是一对重要的协同刺激信号分子,阻断该信号通路可使T细胞处于无反应性的失能状态,诱导免疫耐受。目的:为获得更为理想的免疫耐受状态,检测甲氧基聚乙二醇-琥珀酰亚胺丙酸酸酯(methoxy-polyethyleneglycol-succinimidyl-propionicacidester,mPEG-SPA)化学修饰联合抗OX40L单抗对移植物T淋巴细胞增殖、表型及分泌细胞因子的影响。设计、时间及地点:对比观察移植物免疫耐受体外实验,于2006-12/2007-06在徐州医学院附属医院血液病研究室完成。材料:清洁级近交系C57BL/6(H-2b)雄性和BALB/c(H-2d)雌性成年小鼠各12只,制备的脾淋巴细胞分别作为反应细胞和刺激细胞。mPEG-SPA为北京凯正公司产品,大鼠抗小鼠OX40L单抗为eBioscience公司产品。方法:将反应细胞密度调整为4×109L-1,分别加入3,6,12,15,18g/LmPEG-SPA修饰1h,以加入相同体积的磷酸盐缓冲液作空白对照;另向反应细胞中加入15g/LmPEG-SPA分别修饰1h,24h,48h,96h,120h,流式细胞仪检测脾淋巴细胞表面CD3分子的表达。单向混合淋巴细胞培养分4组:细胞对照组,单纯加入刺激细胞+反应细胞;抗OX40L单抗组,向两种细胞中加入10mg/L抗OX40L单抗;mPEG-SPA组,向两种细胞中加入15g/LmPEG-SPA;联合组,向两种细胞中加入抗OX40L单抗和mPEG-SPA。主要观察指标:CD3分子的表达水平,其反映mPEG-SPA遮蔽脾淋巴细胞的效果。淋巴细胞增殖情况与表型分析。培养上清细胞因子含量。结果:①不同浓度mPEG-SPA修饰后淋巴细胞表面CD3分子的表达均明显低于空白对照,且15,18g/LmPEG-SPA降低幅度尤为明显(P<0.05);15g/LmPEG-SPA修饰不同时间后CD3分子的表达无变化(F=1.715,P>0.05)。与细胞对照组比较,抗OX40L单抗组、mPEG-SPA组、联合组对淋巴细胞的增殖抑制率均明显升高,联合组抑制效果最强(P<0.01);抗OX40L单抗组、BACKGROUND： Methoxy-polyethylene glycol （mPEG） can have a stereospecific blockade, which can shield surface antigen of T lymphocytes. OX40 and its ligand OX40L is a pair of important costimulator. To block the signal pathway can induce T cells in an inexcitahle incapacitation, resulting in immune tolerance. OBJECTIVE： To investigate the effect of combined usage of methoxy-polyethylene glycol-succinimidyl-propionic acid ester （mPEG-SPA） and anti-OX40L monoclonal antibody （McAb） on proliferation, phenotypes of T lymphocytes and secretion of cytokines in Vitro to obtain an ideal immune tolerance. DESIGN, TIME AND SETTING： The in vitro control experiment was performed at the Department of Hematology, Affiliated Hospital of Xuzhou Medical College from December 2006 to June 2007. MATERIALS： Totally 12 clean inbred line C57BL/6（H-2^b） male and BALB/c（H-2^d） female adult mice each were selected. Prepared splenic lymphocytes were used as responder cells and stimulator cells, mPEG-SPA and rat anti-mouse OX40L monoclonal antibody were respectively purchased from Kaizheng, China and eBioscience, USA. METHODS： Responder cells were regulated to a density of 4×10^9 L^-1, and modified by 3, 6, 12, 15, 18 g/L mPEG-SPA for 1 hour. Blank control cells were treated with an equal volume of phosphate buffer saline. In addition, responder cells were given 15 g/L mPEG-SPA for 1, 24, 48, 96 and 120 hours. Expression of CD3^＋ T cells was detected by flow cytometer. In one-way mixed lymphocyte culture, cells in the cell control group received stimulator cells and responder cells. Cells in the anti-OX40L McAb group underwent 10 mg/L anti-OX40L McAb. Cells in the mPEG-SPA group were subjected to 15 g/L mPEG-SPA. Cells in the combination group were treated with anti-OX40L McAb and mPEG-SPA. MAIN OUTCOME MEASURES： CD3 expression; Outcome of mPEG-SPA screening splenic lymphocytes; Proliferation and phenotype of lymphocytes; Cytokine content in supernatant. RESULTS： After modification of mPEG-SPA, expre

关 键 词：甲氧基聚乙二醇 化学修饰 抗原 OX40/OX40L免疫耐受 

分 类 号：R394.2[医药卫生—医学遗传学]