抗体修饰肿瘤细胞疫苗实验研究

发布时间：2018-01-10 21:29

本文关键词：抗体修饰肿瘤细胞疫苗实验研究　出处：《中国协和医科大学》2008年博士论文　论文类型：学位论文

【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。
[Abstract]:Tumor vaccine has been widely studied as one of biotherapy, especially in preventing tumor recurrence and metastasis. Tumor cell vaccine contains all tumor related antigens. So up to now tumor cell vaccine still has irreplaceable advantage. In order to improve the immune effect of tumor vaccine, a new antibody modified tumor cell vaccine has been studied. The mouse model was established and the antigen-antibody binding spatial structure was analyzed by literature review. The cloned human CD20 extracellular and transmembrane region genes were spliced with the secondary lymphoid tissue chemokine signal peptide region. The eukaryotic expression plasmid of fusion gene, pcDNA3f-sig-CD20, was constructed, and the stable clone strain was screened after transfection into B16F10 tumor cells. Flow cytometry and fluorescence microscopy showed that the cell line B16 / CD20 expressed the target protein. Rituxima, a clinical monoclonal antibody against CD20, was detected by Rituximab. After binding, the vaccine was prepared. The model of artificial lung tumor metastasis in the same C57BL / 6 mice was treated and the mechanism of its antitumor effect was discussed. Anti-tumor specific T cells were induced by Rituximab modified human CD20 ~ tumor cells. The anti-HLA-A0201 antibody blocking test was used to verify the anti-tumor effect of MHC. The results showed that the vaccine could effectively interfere with the growth of tumor nodules and prolong the survival time of mice in the experimental animal model of artificial lung metastasis. The results of anti-tumor effect and LDH release assay showed that the vaccine could significantly improve the ability of spleen lymphocytes to kill tumor after immunizing mice. Further verification of CD8 ~ by cell and NK deletion assay. T-lymphocytes are the main effector cells in the process of anti-tumor metastasis. We also observed that the antibody-modified tumor vaccine can not only induce the killing effect of tumor cells transfected with CD20. Confocal microscopy and flow cytometry showed that DC could significantly enhance DC capture rate of tumor cells after incubating with DC. These results suggest that the vaccine enhances the capture of tumor antigen through the FC receptor pathway of antigen-presenting cells. Thus, the antigen presentation effect and the specific anti-tumor effect were improved. The human body external experiment system showed that:. Rituximab modified human tumor cell vaccine induced anti-tumor effector cell. HLA-A2 antibody blocking assay further confirmed that the effector cell was MHC restricted cytotoxic T cell. The preparation method of antibody modified tumor vaccine was simple. It is safe and effective to use in vivo, so the preparation cost is low. This method opens up a new method for clinical anti-tumor monoclonal antibody drugs, and is expected to become a new method of clinical tumor immunotherapy.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392;R730.5

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