链亲和素标记的白细胞介素4双功能融合蛋白锚定治疗小鼠浅表性膀胱癌  

lntravesical anchoring of streptavidin-tagged interleukin-4 fusion protein for immunotherapy of mouse superficial bladder cancer

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

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

出　　处：《中华肿瘤杂志》2012年第5期331-335,共5页Chinese Journal of Oncology

基　　金：国家“863”计划项目（2006AA0224C4）;浙江省自然科学基金（R2080407）

摘　　要：目的探讨链亲和素标记的白细胞介素4（IL-4-SA）双功能融合蛋白治疗小鼠浅表性膀胱癌的效果。方法制备IL-4-SA双功能融合蛋白，测定其体外生物学活性。建立小鼠浅表性膀胱癌模型，膀胱经生物素化后给予IL4-SA灌注治疗，观察小鼠生存期，并以链亲和素标记的绿色荧光蛋白（GFP—SA）＋IL-4和磷酸盐缓冲液（PBS）作为对照组。检测各组小鼠脾细胞的细胞毒性T淋巴细胞（CTL）杀伤活性和CD8＋肿瘤浸润淋巴细胞。结果IL4．SA能够锚定于生物素化的膀胱表面达5d以上。在MIM9细胞种植后的第80天，PBS组小鼠全部死亡，GFP-SA＋IL-4组小鼠存活2只，而IL4-SA组小鼠存活5只。在肿瘤特异性杀伤实验中，在效靶比为1：1、25：1、50：1时，PBS组小鼠的CTL杀伤率分别为（3．3±0．6）％、（7．3±0．6）％和（12．7±2．1）％，GFP．SA＋IL4组小鼠的CTL杀伤率分别为（4．3±0．6）％、（9．0±1．0）％和（14．3±1．5）％，而IL4-sA组小鼠的CTL杀伤率分别为（11．3±1．2）％、（22．7±1．5）％和（31．0±3．0）％，IL4-SA组与PBS组和GFP—SA＋IL-4组比较，差异有统计学意义（P〈0．05）。CD8＋淋巴细胞检测结果显示，PBS组和GFP．SA＋IL4组的肿瘤组织内仅有少量的CD8＋淋巴细胞浸润，而IL-4-SA组则有较多的CD8＋淋巴细胞浸润。结论IL4-SA锚定膀胱表面能够产生强烈、持久的免疫应答反应，有可能成为浅表性膀胱癌的一种新灌注治疗方法。Objective To evaluate the antitumor efficacy of streptavidin-tagged interleukin-4 （IL-4- SA） bifunctional fusion protein in the immunotherapy of mouse model of superficial bladder cancer. Methods IL-4-SA fusion protein was prepared and its biological activity was determined. One day after MB49 cell implantation, 100 μl of 1 mg/ml NHS-PE（M-biotin was instilled into the bladder for 30 minutes, followed by intravesical instillation of 100μl PBS, GFP-SA ＋ IL-4 or IL-4-SA and incubation for 1 hour. The bladder irrigation was performed twice a week for three weeks. The CTL cytotoxicity and profile of CD8＋ tumor-infiltrating lymphocytes were analyzed. Results The IL-4-SA fusion protein was durably anchored to the biotinylated mucosal surface of bladder wall for up to 5 days. On day 80 after the implantation of MB49 cells, all of PBS-treated mice died, and 8 out of 10 mice in the GFP-SA-treated group died from tumor burden. In contrast, 5 out of 10 mice in the IL-d-SA-treated group were tumor-free. The MB49 tumor- specific cytotoxicity from mice in the IL-4-SA group was （11.3± 1.2）%, （22.7 ± 1.5 ）% and （31.0 ± 3. 0） % at the effector to target ratios of 1 ： 1, 25 ： 1 and 50 ： 1, respectively. But the corresponding cytotoxicity was （4.3 ±0. 6） %, （9.0±1.0） % and （ 14.3 ± 1.5 ） % in the GFP-SA ＋ IL-4 group, and （3.3±0.6）%, （7.3±0.6）%, （12.7 ± 2.1 ）% in the PBS group. The tumor-specific cytotoxicity in the SA-CIMOL group was significantly higher than that in the control groups （P 〈 0.05）. The infiltrating CD8＋T cells in tumors in the IL-4-SA-treated group were increased compare with those in other groups. Conclusion Intravesical anchoring of IL-4-SA elicites strong and long-lasting immunoprotection against superficial bladder cancer, and the novel immunotherapy may be an attractive therapeutic alternative in future.

关 键 词：膀胱肿瘤 白细胞介素4 链亲和素 融合蛋白 小鼠 

分 类 号：R737.14[医药卫生—肿瘤]