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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 the streptavidin-tagged interleukin-7 (SA-IL-7) fusion protein and evaluate its effect on the treatment of superficial bladder cancer in mice. Methods: The recombinant plasmid pET24a-SA-IL-7 was constructed and SA-IL-7 fusion protein was prepared. The binding of SA-IL-7 fusion protein to biotinylated bladder cancer cell line MB49 was detected by flow cytometry. The biological activity of SA-IL-7 fusion protein was detected by thymocyte proliferation assay to promote thymocyte proliferation. The effect of intravesical instillation of SA-IL-7 was evaluated by using mouse superficial bladder cancer model. The survival of mice was observed. The tumor infiltrating CD8 + T cells were detected by immunohistochemistry. Results: The successfully prepared SA-IL-7 fusion protein could be anchored on the surface of biotinylated MB49 cells with the binding rate of (96.6 ± 1.3)%; at the same time, it can promote the proliferation of thymocytes. The SA-IL-7 fusion protein could be anchored to the biotinylated bladder mucosal surface of mice for more than 7 days, while the non-biotinylated bladder mucosa was never detected for the presence of IL-7. In the mouse superficial bladder cancer model, 90% of the mice in the IL-7 perfusion treatment group died on the 80th day after the inoculation of MB49 cells and 60% of the mice in the SA-IL-7 group survived without tumor (P <0.05). The number of infiltrating CD8 + T cells in SA-IL-7-treated mice increased significantly (P <0.01). Of the 15 superficial bladder cancer model mice that were cured with SA-IL-7, 11 were resistant to a second inoculation of MB49 cells in the bladder compared with only 1 in 15 control mice (P <0.01). Conclusion: Intravesical instillation of SA-IL-7 fusion protein can produce anti-tumor immune response and obvious therapeutic effect, which may become a new method for immunotherapy of superficial bladder cancer.