EGFRvⅢ-scFv/IP-10纳米颗粒促进胶质瘤/DC疫苗抗脑胶质瘤研究

How to improve glioma/DC vaccines anticancer in vivo is a challenge that must be tackled. This subject want to construct EGFRvⅢ-scFv/IP-10 nanoparticles on the basis of the previous work, which has been constructed with IP10 eukaryotic expression vector and EGFRvⅢ-scFv by modified with chitosan nanoparticles. Then we used nanoparticles to transfect normal cells and cancer cells, to make relative research in vitro. Then, we estabulished murine brain glioma model and imported nanoparticles. Simultaneously, the nanoparticles have ability of specific binding to EGFRvⅢon the surface of cancer cells and improve the concentration of IP-10. Therefore, glioma-specific CTL produced by glioma/DC vaccine were enriched around the tumor cells by IP-10,and enhanced their anticancer activity. At the same time, they formed into multiple of EGFRvⅢ positive cells as the center of the main battlefield of CTL annihhilating the tumor cells. Accordingly, we will study the mechanism of molecular immunology of the nanoparticles reactor furtherly. There is no doubt that it lays a solid theorectical foundation and provide strong experimental evidence for its applying to clinical as soon as possible.

如何提高胶质瘤致敏的树突状细胞（DC）疫苗在体内的抗癌活性是亟待攻克的难题。本课题在前期工作基础上，用已构建好的IP-10基因真核表达载体和抗EGFRvⅢ单链抗体（EGFRvⅢ-scFv）修饰的壳聚糖纳米粒子反应制备成EGFRvⅢ-scFv/IP-10纳米颗粒。将该纳米颗粒转染正常细胞和肿瘤细胞，进一步完善体外实验及其相关研究。应用已建立的小鼠脑胶质瘤模型，将纳米颗粒导入体内通过与肿瘤细胞表面的EGFRvⅢ受体特异结合，使肿瘤部位获得高浓度IP-10，从而通过IP-10将胶质瘤/DC疫苗产生的胶质瘤特异性CTL富集至胶质瘤细胞周围，并增强他们抗癌活性，形成数个以EGFRvⅢ阳性细胞为中心的CTL歼灭肿瘤的主战场，最终实现抗胶质瘤效应。在此基础上，我们将进一步系统研究该反应器发生效应的分子免疫学机制。该研究将为其尽早应用于胶质瘤临床治疗奠定坚实的理论和实验基础。

本课题在前期研究工作基础上，用已经构建好的IP-10基因真核表达载体和抗EGFRvIII的单链抗体EGFRvIII-scFv修饰的壳聚糖纳米粒子反应制备成EGFRvIII-scFv/IP-10纳米颗粒，通过抗原抗体结合实验和趋化实验证实该纳米粒子能与表达EGFRvIII的胶质瘤细胞特异性结合，同时具有趋化淋巴细胞的作用。为提高脑胶质瘤致敏的树突状细胞（DC）疫苗在体内的抗癌活性，我们用淋巴细胞分离液将人脐带血的淋巴细胞分离出来，然后用磁珠分选法分选出淋巴细胞中的DC细胞，与脑胶质瘤裂解肽共同孵育得到胶质瘤/DC疫苗，并与T淋巴细胞共同培养3-4周，得到肿瘤特异性CTL细胞，通过ELISPOT检测法和胞内细胞因子检测法，检测到扩增的CTL对胶质瘤细胞刺激后能大量分泌细胞因子IFN-γ和IL-2，并用51Cr释放法检测到CTL对特异靶细胞具有杀伤效应，而对非靶细胞及其对照细胞无杀伤效应，为进一步证实其体内抑制肿瘤的效果，我们建立了BABL/C/Nude nu裸鼠脑胶质瘤模型，通过尾静脉注射EGFRvIII-scFv/IP10纳米颗粒和胶质瘤DC疫苗，与对照组比较，观察到裸鼠的生存周期明显延长，并将肿瘤取出后，用流式细胞仪检测到肿瘤浸润的T淋巴细胞较不含肿瘤的对侧脑组织明显增多。因此，我们构建的EGFRvIII-scFv/IP-10纳米颗粒不仅能提高胶质瘤/DC 疫苗抗肿瘤活性，而且将先前经颅给药改为静脉给药，为后期应用于临床奠定了坚实的基础。