B7.1/GM-CSF修饰的PC3-DC融合疫苗在体外抗肿瘤免疫效应

Antitumor effect of PC3-DC fusion vaccines modified by B7.1/GM-CSF in vitro

目的:制作B7.1/GM-CSF修饰的DC融合疫苗,并观察其在体外的特异性抗肿瘤免疫效应。方法:制备完整表达GMCSF的前列腺癌细胞(PC3细胞),从前列腺癌患者外周血中分离树实状细胞(DC),应用电融合法融合DC和GM-CSF-PC3,构建B7.1/GPI蛋白并锚定于融合细胞表面;应用免疫荧光观察融合细胞形态及锚定稳定性;流式细胞进行融合细胞表形测定;ELISA试剂盒检测IFN-γ分泌情况;MTT及LDH试剂盒测定融合细胞刺激同源异体T淋巴细胞增殖和细胞毒性效应。结果:成功制备GM-CSF-PC3细胞及B7.1/GPI蛋白,成功分离DC并表达DC分子标记;B7.1/GM-CSF融合疫苗高表达DC分子标记及前列腺癌标记;T细胞增殖实验证明融合疫苗具有强烈的免疫刺激活性;细胞毒性实验证明了融合细胞具有比对照组更有效的肿瘤杀伤作用。结论:电融合技术可成功诱导DC与GM-CSF-PC3融合,体外实验中特定修饰的融合疫苗可显著的提升抗肿瘤免疫效应。

Objective： To construct the fusion vaccine with dendritic cells and prostate cancer cells（PC3） and modify it by B7.1/GM-CSF,and test the anti-tumor immunological reaction of the fusion vaccine in vitro. Methods： Fusion cells was electrofusioned by DC and GMCSF-PC3, and B7.1/GPI protein was constructed and anchored to the fusion cell surface; Cell morphology and anchoring stability were determined by immunofluorescence, and fusion cell phenotype were determined by flow cytometry; fusion vaccines stimulated homologous proliferation and cytotoxic effect of allogeneic T lymphocytes were determined by MTT and LDH kit. Results： B7.1/GM-CSF fusion vaccine highly expressed DC molecular markers and prostate cancer markers; B7.1/GM-CSF fusion vaccines strongly promoted the proliferation of T cells and induced a significantly tumor-specific cytotoxic T lymphocyte（CTL） response and killed the PC3 cells effectively in vitro.Conclusion： The DC and GM-CSF-PC3 can be successfully fused by electrical fusion technique, and the specific modified fusion vaccine can significantly enhance the anti-tumor immune effect in vitro.