小鼠galectin-9可通过分泌形式诱导Tim-3^-T细胞凋亡  

Apoptosis of Tim-3^- T cells induced by secretion of galectin-9 in mice

作　　者：何文涛[1] 袁劲[2] 徐逸[1] 周鸿敏[1] 蔡兰军[1] 郭晖[1] 李超[1] 左利群[1] 宫念樵[1] 陈忠华 

机构地区：[1]华中科技大学同济医学院附属同济医院器官移植研究所,教育部/卫生部重点实验室,武汉430030 [2]华中科技大学同济医学院附属同济医院肾内科

出　　处：《中华微生物学和免疫学杂志》2010年第2期144-149,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30671989,30800524）;国家重点基础研究发展规划（973计划）（2009CB522407） 志谢：感谢Toshiro Niki博士（GalPharma Ltd,Japan）提供pBKC-MV-mG9质粒和大鼠抗小鼠galectin-9单抗,以及Ritter Thomas教授（National University of Ireland,Galway,Ireland）提供腺病毒质粒pENTRlA DualSelection和pAd/CMV/V5-DEST.

摘　　要：目的 构建小鼠galectin-9重组腺病毒pAd-gal-9,探讨galectin-9的真核表达定位以及galectin-9诱导T细胞凋亡与Tim-3表达的关系.方法 常规分子克隆方法 结合LR反应构建腺病毒重组质粒pAd/CMV/V5-DEST-gal-9,以Pac I线性化后,用脂质体2000体外转染293A细胞,8 d后反复冻融细胞3次收集含病毒上清,并以此上清感染293A细胞大量扩增病毒pAd-gal-9.CsCI密度梯度离心法纯化病毒,96孔板法梯度感染293A细胞测定病毒滴度,然后感染CHO细胞,用免疫组化、Western blot和流式细胞仪分析galectin-9的表达水平和定位;以新鲜培养的上清或固相化的细胞分别与外周淋巴结细胞进行培养,AnnexinV/PI法检测T细胞凋亡情况,对比凋亡细胞比例与Tim-3^＋ T细胞的比例.结果 重组腺病毒构建及表达成功,免疫组化表明galectin-9在CHO胞质表达;Westernblot证实galectin-9表达;流式分析表明胞内染色组galectin-9平均荧光强度显著高于表面染色组,病毒感染CHO表面染色组与空白对照组galectin-9平均荧光强度无明显区别;新鲜培养的上清可以明显诱导T细胞发生凋亡,显著高于Tim-3^＋ T细胞的比例.结论 重组腺病毒pAd-gal-9构建成功,体外感染pad-gal-9的CHO分泌galectin-9诱导T细胞凋亡可以不依赖Tim-3的表达.Objective To prepare recombinant adenovirus pAd-gal-9 containing murine galectin-9 and explore galectin-9＇s pro-apoptotic effect on T lymphocytes. Methods The recombinant adenovirus plas-mid pAd/CMV/V5-DEST-gal-9 was prepared by conventional molecular cloning and LR reaction. The pAd/ CMV/V5-DEST-gal-9 linearlized by Pac I was transfected into 293A cells with Lipofectin 2000. Eight days after transfection, the 293A cells were subjected to freeze/thraw circle for three times and the supernatant was collected after centrifugation. Higer titer pAd-gal-9 was produced by large-scale infection of 293A cells with the supernatant containing pAd-gal-9. The supernatant was condensed to get purified pAd-gal-9 by CsCl density gradient centrifugation. After titer determination with gradient dilution of harvested pAd-gal-9 infec-tion in 293A-seeded 96-wells, pAd-gal-9 was used to infect the CHO cell line. Immunohistological assay, Western blot and flow cytometry were employed to ascertain the subcellular location expression of galectin-9. We added solid-phase transgenic CHO cells or freshly-cultured supernatant to medium containing activated T cells to detect the pro-apoptotic effect of galectin-9. Results The pAd-gal-9 was prepared successful. Im-munohistochemical staining of CHO infected with pAd-gal-9 confirmed that galectin-9 was expressed in the cytosol. Intercellular staining indicated that mean fluorescence intensity of galectin-9 was significantly higher in pAd-gal-9-infected CHO group than control group. Supernatant from pAd-gal-9-infected CHO promoted the apoptosis of T cells. The percent of apoptotic T cells was higher than the Tim-3 positive T cells. Conclu-sion CHO infected with pAd-gal-9 can secret galectin-9 to promote the apoptosis of activated T cells via Tim-3-independent mechanisms.

关 键 词：半乳糖凝集素-9 胞内染色 凋亡 T细胞 

分 类 号：R363[医药卫生—病理学]