粉尘螨抗原免疫治疗小鼠结肠癌的初步研究-生物化学与分子生物学专业论文.docxVIP

摘要 摘要 万方数据 万方数据 摘 要 结肠癌是最常见的恶性肿瘤之一，其发病率和死亡率居高不下，全球每年 有 120 万例新增结肠癌患者，目前对结肠癌的治疗仍然以手术、放疗和化疗为 主。我们研究发现结肠癌患者中 Treg 细胞比例较正常人高，而在过敏性疾病患 者中 Treg 细胞比例较正常人低。本研究使用粉尘螨粗蛋白和 Der f2 重组蛋白用 于治疗小鼠结肠癌模型以及对治疗效果进行一系列检测。 方法：首先制备和鉴定粉尘螨粗蛋白和表达纯化 Der f2 重组蛋白，摸索建 立合适的小鼠结肠癌模型；制定免疫治疗方案后进行建模及治疗，监测小鼠肿 瘤生长状况；处死小鼠后，对小鼠肿瘤进行称重。用流式细胞的方法检测 CD8+T 细胞比例及 CD4+CD25+Foxp3+Treg 细胞比例；用荧光定量 PCR 的方法检测 Foxp3 转录水平；分别用 Western-blot 印迹杂交和免疫组化检测小鼠肿瘤组织内 Foxp3 蛋白水平。 结果：摸索和建立了合适的小鼠结肠癌模型；成功制备和鉴定了粉尘螨粗 蛋白以及表达纯化的 Der f2 重组蛋白，并用于小鼠结肠癌模型的治疗；经过治 疗后，治疗组小鼠肿瘤生长速度均减缓，其肿瘤重量均低于未治疗组；经流式 细胞检测，治疗组小鼠体内 CD8+T 细胞比例上升，CD4+CD25+Foxp3+Treg 细 胞比例下降，与未治疗组相比，治疗组小鼠体内 Foxp3 转录水平均明显减低； Western-blot 和免疫组化检测结果显示治疗组小鼠肿瘤中 Foxp3 蛋白水平均降 低。 结论：本实验结果表明粉尘螨粗蛋白和 Der f2 重组蛋白均能有效治疗小鼠 结肠癌模型，但其分子机理有待进一步实验证明。用粉尘螨抗原治疗结肠癌有 望成为癌症的免疫治疗新方法。 关键词：结肠癌；粉尘螨；Treg 细胞；Foxp3 II Ab Abstract ABSTRACT Colon cancer is one of the most common malignant tumors, the levels of incidence and mortality of colon cancer remain high. There are 1.2 million patients were diagnosed as colon cancer each year in the world. The most popular treatments of colon cancer are surgery, radiotherapy and chemotherapy. Research finds that there are higher rate of regulatory T (Treg) cells in colon cancer patients than in normal persons, and a contrary situation shows less Tregs in allergic disease patients than that in normal persons. In this study, we used dust mite crude proteins and recombinant protein Der f2 to treat a Balb/c mice model of colon cancer and the treatment effects were evaluated Methods: First of all, we prepared dust mite crude proteins and a recombinant Der f2 protein. A Balb/c mouse model of colon cancer was induced. Afterwards, and the mice were treatedwith the dust mite crude proteins or recombinant protein Der f2. The growth of the tumors was monitored. After sacrifice, the tumors were weighed. The frequency of CD8+T cells and CD4+CD25+ Foxp3+Treg cells in the tumor tissue was assessed by flow cytometry . The gene transcriptional levels of Foxp3 were analyzed by quantitative RT-PC