【作者】 王辉丽；

【导师】 张叔人；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2008， 博士

【摘要】 肿瘤疫苗作为生物疗法之一被人们广泛研究,尤其在防止肿瘤复发转移中具有重要价值。肿瘤细胞疫苗含有所有的肿瘤相关抗原,所以至今肿瘤细胞疫苗仍有不可替代的优势。为了提高肿瘤疫苗的免疫效果,该课题研究了新型的抗体修饰肿瘤细胞疫苗。为了进行临床前的体内实验研究,我们建立了小鼠模型。通过文献查阅和抗原抗体结合空间结构分析,选择克隆人CD20胞外区和部分跨膜区基因与次级淋巴组织趋化因子信号肽基因区域进行拼接,构建了融合基因真核表达质粒(pcDNA3f-sig-CD20)。该质粒导入B16F10肿瘤细胞后筛选出稳定克隆株,经流式细胞法和荧光显微镜验证该细胞株(B16/CD20)表达目的蛋白。通过与临床抗CD20单克隆抗体药物Rituximab(美罗华)结合后制备成疫苗,治疗同系C57BL/6小鼠人工肺肿瘤转移模型,并对其抗肿瘤效应机制进行了初步探讨。此外,应用Rituximab修饰人CD20~+肿瘤细胞制备疫苗诱导抗肿瘤特异性T细胞,并用抗HLA-A0201抗体封闭实验验证MHC限制性的杀伤肿瘤作用。研究结果显示,在人工肺转移肿瘤实验动物模型中,该疫苗能有效的干预肿瘤结节的生长,延长小鼠生存期,获得较好的抗肿瘤效果。LDH释放法测定表明:该疫苗免疫小鼠后显著提高了脾淋巴细胞特异性杀伤肿瘤的能力。CD8~+细胞和NK删除实验进一步验证CD8~+T淋巴细胞在抗肿瘤转移过程中是主要的效应细胞。实验中我们还观察到抗体修饰瘤苗不仅能诱导针对转染了CD20的肿瘤细胞杀伤反应,而且还提高对野生型黑色素瘤的杀伤效应。共聚焦显微镜和流式细胞测定显示:抗体修饰瘤苗与DC孵育后能明显增强DC对肿瘤细胞的捕获率,提示该疫苗通过抗原呈递细胞Fc受体途径增强对肿瘤抗原捕捉,从而提高了抗原呈递效果和诱导了特异性抗肿瘤作用。人体外实验系统表明:Rituximab修饰人的肿瘤细胞疫苗诱导了抗肿瘤效应细胞。HLA-A2抗体封闭实验进一步证实了该效应细胞是MHC限制性的细胞毒性T细胞。我们建立的抗体修饰瘤苗制备方法简单,体内使用安全有效,抗体用量较少,因此制备成本低廉。此法为临床抗肿瘤单克隆抗体药物开辟了新的应用方法,有望成为临床肿瘤免疫治疗的新手段。更多还原

【Abstract】 Our study is to clone the extracellular and transmembrane domains (ECD and TMD) of human CD20 and construct its eukaryotic expression vector for the evaluation of the possability of antibody coated tumor cell vaccine therapy in animal model. The desired result is to provide antigens in a stimulatory immunotherapy which allows the in vivo generation of a potent anti-tumor immune response.The whole cDNA was amplified by RT-PCR method from the Raji cells and cloned to the downstream of expression vector pcDNA 3f-sig with a signal pepetide. The fragment was identified by enzyme digestion and DNA sequencing. Cells stably expressing sig-CD20 were established by screening with G418 after transfection and characterization of the transfected B16F10 cell line were rightly evaluated by indirect immunoflurescence assay and FACS. To further explore the rituximab mediated antitumor effect, a Rituximab modified tumor cell vaccine which had been inactivated by mitomycin C(MMC)was developed and treated in its syngeneic C57BL/6 mice model effectively. The results demonstrated that the Rituximab-coated tumor cell vaccine had a significant therapeutic effect against tumor pulmonary metastasis formation. The CTLs activity was remarkably higher from the mice vaccinated by Rituximab-coated tumor cell vaccine than normal Ig-coated tumor cell vaccine. Capture rate of tumor cells by DCs, which were detected by flow cytometry, was increased by adding Rituximab. The tumor specific cytolysis could be induced by Rituximab-coated tumor cell in human in vitro assay. Human tumor specific CTLs could also be highly induced by Rituximab-coated tumor cell vaccine. It was inhibited by anti- HLA-A2 site monoclonal antibody. This therapeutic strategy provides a simple way to potentialize CTLs function to combat cancer and may promote more clinical consideration in immunotherapy for tumors. Rituximab-coated tumor cell vaccine also expanded the clinical Rituximab applications.更多还原