SA-hTNF-α膜锚定修饰治疗表浅膀胱癌的实验研究  

Immobilization of streptavidin-tagged bioactive hTNF-α on biotinylated mucosal surface of the bladder wall for treatment of superficial bladder cancer in mice

作　　者：陈忠[1] 谭万龙[1] 黄鑫[2] 梁中锟[1] 徐翠香[2] 高基民[2] 

机构地区：[1]南方医科大学南方医院泌尿外科,广东广州510515 [2]南方医科大学生物技术学院生物治疗研究所,广东广州510515

出　　处：《南方医科大学学报》2010年第5期936-940,共5页Journal of Southern Medical University

基　　金：国家863高技术研究发展计划(2006AA02Z4C4);广东省自然科学基金(9151051501000030)

摘　　要：目的探讨一种新型蛋白质锚定技术与人肿瘤坏死因子α(hTNF-α)相结合在小鼠表浅膀胱癌治疗中的作用。方法将120只雌性C57BL/6j小鼠分为5组:正常未处理组(空白对照组)、PBS对照组、可溶hTNF-α治疗组、SA-GFP锚定组、SA-hTNF-α锚定治疗组,每组22只小鼠。建立小鼠正位表浅膀胱癌模型,24h后,将小鼠膀胱内膜生物素化,继而将hTNF-α融合蛋白灌注入小鼠膀胱中。每4d重复膀胱灌注锚定治疗1次,共6次。免疫组化检测SA-hTNF-α融合蛋白在生物素化的小鼠膀胱黏膜的存留时间及膀胱黏膜和肿瘤组织中CD4+T淋巴细胞及CD8+T淋巴细胞的分布,观察肿瘤生长情况及小鼠的生存期。检测肿瘤特异性淋巴细胞的杀伤活性。用MB49细胞再次攻击对SA-hTNF-α融合蛋白治疗有效的小鼠,观察肿瘤生长情况及小鼠的存活期。结果免疫组化显示:SA-hTNF-α融合蛋白可以在膀胱黏膜表面上稳定存留7d;MB49肿瘤细胞膀胱内种植后第60天,SA-hTNF-α锚定治疗组有18只小鼠存活(18/22),其中9只小鼠体外无触及肿瘤;PBS组全部死亡。对9只无瘤存活的小鼠再次用MB49细胞皮下攻击,60d后5只小鼠仍存活(5/9),与对照组有显著性差异(P<0.05)。膀胱癌病灶中CD4+T淋巴细胞及CD8+T淋巴细胞,SA-hTNF-α锚定治疗组较PBS对照组明显增多(P<0.05)。SA-hTNF-α锚定治疗组小鼠的杀伤效应明显强于正常组小鼠(P<0.05)。结论 SA-hTNF-α稳定的原位锚定于小鼠膀胱黏膜表面,可有效抑制小鼠表浅膀胱癌进展,显著延长荷瘤小鼠的生存时间,有效抵抗同源肿瘤细胞的再次攻击。Objective To investigate a novel immunotherapy through immobilization of streptavidin-tagged hTNF-α on the biotinylated mucosal surface of the bladder wall for bladder cancer treatment in mice. Methods A total of 120 female C57BL/6j mice were randomized into 5 equal groups, namely blank control, PBS, soluble hTNF-α, SA-GFP, and SA-hTNF-α treatment groups. Twenty-four hours after establishment of a mouse model of orthotopic superficial bladder cancer, SA-hTNF-α fusion protein was immobilized on the biotinylated mucosal surface of the bladder wall, which was repeated every 4 days for a total of 6 sessions. Immunohistochemistry was performed to detect the retention time of SA-hTNF-α fusion protein in the biotinylated mouse bladder mucosa and the distribution of CD4＋ and CD8＋ lymphocytes in the mucosa and tumor tissues, with the tumor growth and mouse survival also observed. The cytotoxiciy of the tumor-specific lymphocytes was evaluated. The mice responding well to the treatment were re-challenged by MB49 and monitored for survival. Results SA-hTNF-α could be efficiently and stably immobilized on the bladder mucosal surface for as long as 7 days. On day 60 after MB49 implantation, 18 out of 22 SAhTNF-α-treated mice survived, with 9 appearing tumor-free, but all the mice in PBS control group died. Five out of 9 tumor-free mice in SA-hTNF-α group showed resistance to a re-challenge with intravesical MB49. The numbers of CD4＋ and CD8＋ lymphocytes were significantly greater in SA-hTNF-α group than in the other groups （P0.05）. The cytotoxicity of the tumor-specific lymphocytes was significantly stronger in SA-hTNF-α group than in the other groups （P0.05）. Conclusion SA-hTNF-α immobilized on the biotinylated mucosal surface of the bladder wall can significantly inhibit the tumor growth and promote the survival of the mice bearing orthotopic superficial bladder cancer.

关 键 词：肿瘤坏死因子Α 链亲和素 生物免疫治疗 膀胱癌 

分 类 号：R737.14[医药卫生—肿瘤]