SA-IL-7融合蛋白的制备及其膀胱内灌注对小鼠表浅性膀胱癌的治疗效应  

Preparation of streptavidin-tagged interleukin-7 fusion protein and its therapeutic effect with intravascal instillation on mouse superficial bladder cancer

作　　者：张振[1] 贺利民[1] 马磊[1] 李金龙[2] 许晓玲[3] 高基民[3] 

机构地区：[1]南阳市第一人民医院肿瘤科,河南南阳473000 [2]南方医科大学生物治疗研究所,广东广州510515 [3]温州医科大学浙江省模式生物技术与应用重点实验室,浙江温州325035

出　　处：《中国肿瘤生物治疗杂志》2013年第5期529-534,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家863计划基金资助项目(No.2006AA02Z4C4);浙江省自然科学基金资助项目(No.R2080407)~~

摘　　要：目的:制备链亲和素标记的白介素7(streptavidin-tagged interleukin-7,SA-IL-7)融合蛋白,评价其对小鼠表浅性膀胱癌治疗的效果。方法:构建pET24a-SA-IL-7重组质粒,制备SA-IL-7融合蛋白。流式细胞术检测SA-IL-7融合蛋白与生物素化膀胱癌细胞MB49的结合,胸腺细胞增殖法检测SA-IL-7融合蛋白促进胸腺细胞增殖的生物学活性。应用小鼠表浅性膀胱癌模型评价膀胱内灌注SA-IL-7的治疗效果,观察小鼠生存期,免疫组化法检测各组小鼠肿瘤浸润CD8+T细胞。结果:成功制备的SA-IL-7融合蛋白可锚定于生物素化的MB49细胞表面,结合率达(96.6±1.3)%;同时能够促进胸腺细胞的增殖。SA-IL-7融合蛋白能够锚定于小鼠生物素化的膀胱黏膜表面7 d以上,而未生物素化的膀胱黏膜在始终未检测到IL-7的存在。在小鼠表浅型膀胱癌模型中,MB49细胞接种后的第80天,IL-7灌注治疗组90%小鼠死亡,而SA-IL-7组60%小鼠存活且未出现肿瘤(P<0.05);且SA-IL-7灌注治疗组小鼠肿瘤组织中浸润CD8+T细胞明显增多(P<0.01)。SA-IL-7治愈的15只表浅型膀胱癌模型小鼠中,11只能够抵抗膀胱内MB49细胞的第二次接种,而对照组15只小鼠仅1只幸存(P<0.01)。结论:SA-IL-7融合蛋白膀胱内灌注能够产生抗肿瘤免疫应答和明显的治疗效果,有可能成为免疫治疗浅表性膀胱癌的新方法。Objective ： To prepare streptavidin-tagged interleukin-7 （SA-IL-7） fusion protein and to value its therapeutic effect on mouse superficial bladder cancer. Methods： Recombinant pET24a-SA-IL-7 plasmid was constructed and then SA-IL-7 fusion protein was prepared. The binding of SA-IL-7 to biotinylated bladder cancer MB49 cells was examined by flow cytometry and the bioactivity of SA-IL-7 fusion protein was examined by thymocyte proliferation assay. The mouse superficial bladder cancer model was used to evaluate the therapeutic effect of intravesical instillation of SA-IL-7. Mice were monitored for survival and the tumor-infiltrated CD8＋T cells were examined by immunohistochemistry assay. Results： SA-IL-7 fusion protein efficiently anchored on the biotinylated MB49 cell surface, with a binding ratio of （96.6±13）%; SA-IL-7 promoted the proliferation of thymocytes. The SA-IL-7 fusion protein anchored on biotinylated bladder mucosal surface for more than 7 d. Whereas, IL-7 was no longer detected in the first day in the unbiotinylation group. After being attacked by bladder cancer MB49 cells for 80 d, 90% mice in the IL-7 group, while 60% mice survived without tumors in the SA-IL-7 group （P〈0.05）. Moreover, tumor-infiltrated CD8＋ T cells in the SA-IL-7 group was significantly higher than those in the control group （P〈0.01）. Importantly, 11 out of 15 SA-IL-7-cured mice resisted to a second intravesical MB49 cell challenge, whereas only 1 of 15 control mice survived the challenge （P〈0.01）. Conclusion： Intravesical instillation of SA-IL-7 fusion protein can induce anticancer immunity, which may represent a promising immunotherapy for superficial bladder cancer.

关 键 词：白细胞介素7 链亲和素 生物素 融合蛋白 表浅性膀胱癌 

分 类 号：R737.14[医药卫生—肿瘤] R730.54[医药卫生—临床医学]