可诱导共刺激分子与人IgGFc融合蛋白诱导同种免疫低应答的体内外实验  

Experimental study on induction of allo-hyporesponsiveness by ICOS-Ig in vitro and in vivo

作　　者：张鹏[1] 秦琴[1] 王振猛[2] 沈茜[1] 

机构地区：[1]第二军医大学附属长海医院检验科,上海200433 [2]第二军医大学附属东方肝胆外科医院麻醉科

出　　处：《中华器官移植杂志》2009年第7期389-393,共5页Chinese Journal of Organ Transplantation

基　　金：国家高技术研究发展计划（“863”计划）（2002AA214091）;国家自然科学青年基金（30500501）

摘　　要：目的探讨真核表达人可诱导共刺激分子（ICOS）与人IgGFc融合蛋白（ICOS-Ig融合蛋白）在体内外对同种免疫应答的影响。方法构建ICOS-Ig融合蛋白表达载体，在CHO细胞中表达并纯化ICOS-Ig融合蛋白。以Balb／c小鼠脾细胞为反应细胞，经CO^60照射灭活的C57BL／6小鼠脾细胞为刺激细胞，进行初次混合淋巴细胞反应（MLR），MLR体系中分别加入50μg／ml ICOS-Ig融合蛋白（ICOS-Ig组）或对照IgG（IgG组），采用氚标记胸腺嘧啶脱氧核苷（^3H-TdR）掺入法检测反应细胞的增殖情况，酶联免疫吸附试验（ELISA）检测培养上清液中白细胞介素（IL）2、4、10以及γ干扰素（IFN-γ）的含量。收集初次MLR细胞，与灭活的C57BL／6小鼠脾细胞或C3H小鼠脾细胞共培养，进行再次MLR，检测指标同初次MLR。以Co^60照射Balb／c小鼠，经尾静脉输注用羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）标记的C578126小鼠脾细胞，每天腹腔注射ICOS-Ig融合蛋白0．2mg（ICOS-Ig组）、IgG（对照IgG组）或环孢素A（CsA组），3d后取Balb／c小鼠脾细胞，流式细胞仪测定CFSE荧光强度以判断同种T淋巴细胞的体内增殖情况。结果初次MLR显示，ICOS-Ig组同种T淋巴细胞活化增殖的抑制率为（58±8）％，其培养上清液中IFN-γ的水平明显高于IgG组（P〈0．05）。再次MLR显示，ICOS-Ig融合蛋白能特异性抑制C57BL／6小鼠脾细胞所致的细胞增殖，抑制率为（42±8）％，IL-4和IL-10的分泌受到抑制，而IFN-γ的分泌增加；ICOS-Ig融合蛋白并不抑制第三方细胞所致的细胞增殖。体内实验显示，ICOS-Ig组和CsA组的CFSE荧光强度明显强于空白对照组和对照IgG组（P〈0．05），而联合处理组CFSE荧光强度强于ICOS-Ig组和CsA组（P〈0．05）。结论ICOS-Ig融合蛋白在体内外均可抑制同种T淋巴细胞的活化增殖，且这种作用具有特异性。Objective To express human inducible costimulator （ICOS） extracellular region and IgG Fc fusion protein, and analyze their function in allogenic lymphocyte proliferation in vitro and in vivo. Methods Human ICOS extracellular region and IgG Fc fragment were cloned into a soluble expression vector. ICOS-Ig fusion protein was expressed and purified in CHO cells. To monitor primary MLR, Balb/c spleen T cells were isolated as responder cells, and irradiated C57BL/6 spleen cells as stimulator cells. 50 μg/ml ICOS-Ig or IgG was added to primary MLR cultures. The cells responsive rates were detected by 3 H-TdR methods. ELISA tested supernatants for cytokines （IL2, IL-4, IL-10 and IFN-γ）. T cells of each group in primary MLR were cultured as responder cells for secondary MLR, and irradiated C57BL/6 （donor） or C3H （third party） spleen cells as stimulator cells. Similar indexes were detected in secondary MLR. Then vital dye CFSE was used to study allo reactive T cell proliferation in vivo. CFSE-labeled C57BL/6 spleen cells were transferred to irradiated Balb/c mice. Mice were then intraperitoneally injected with 0. 2 mg IgG, ICOS-Ig or CsA each day.At the 3rd day after transfection, the spleen cells of the mice were harvested to detect CD4＋ CFSE＋ and CD8＋ CFSE＋ by FACS. Results In primary MLR, ICOS-Ig inhibited allogenic T-cell proliferation with inhibition rate being （58 ± 8）% in 50μg/ml, and increased IFN-γ, secretion. In secondary MLR, ICOS-Ig specifically inhibited the proliferation of donor spleen cells with inhibition rate being （42 ± 8） %, and in ICOS-Ig group the levels of IL-4 and IL-10 were lower and the level of IFN-γhigher than in IgG group. However, ICOS-Ig didn＇t inhibit the proliferation of third-party spleen cells. In the CFSE dye assay, CFSE intensity of CD4^＋ and CD8^＋ T cells in ICOS-Ig and CsA groups was stronger than that in control group （P〈 0. 05）, while CFSE intensity in combined treatment group were even stronger than that in ICOS-Ig and CsA groups

关 键 词：抗原 CD28 免疫球蛋白G 重组融合蛋白质类 免疫抑制法 淋巴细胞培养试验 混合 

分 类 号：R686[医药卫生—骨科学]