论文部分内容阅读
目的探讨以改构腺相关病毒2型(AAV-2)为载体,将rAAV/CEA转染树突状细胞(Dendritic cell,DC)制备肺癌治疗性疫苗的可行性。方法取健康人外周血,分离获得贴壁的单个核细胞(Mo),以rAAV/CEA病毒转染,对照组以CEA多肽刺激,两组细胞均以重组人粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4(IL-4)、肿瘤坏死因子-α(TNF-α)诱导DC成熟。第7天收集DC,取DC与原始T细胞混合,诱导细胞毒性T细胞(CTL),[3H]-TdR掺入法检测DC刺激自体T细胞增殖能力;流式细胞仪分析CTL细胞中IL-4、IFN-γ、CD8、CD4、CD69、CD25的表达情况;取CEA阳性的肺癌细胞株A549为靶细胞,采用51Cr释放法检测CTL对该靶细胞的杀伤效率。结果 rAAV/CEA转染的DC疫苗可诱导特异性细胞免疫,具体表现在:⑧疫苗具备较强的刺激淋巴细胞增殖的能力,⑧诱导的CTL较高表达CD8、CD69和IFN-γ,⑧对CEA阳性的肺癌靶细胞产生较强杀伤活性,且此活性具有抗原特异性和MHC-I类限制性。结论以AAV为载体,rAAV/CEA基因转染DC可成功制备针对肺癌的治疗性疫苗。
Objective To investigate the feasibility of using rAAV / CEA to transfect dendritic cells (DCs) as a therapeutic vaccine for lung cancer using AAV-2 as a vector. Methods Adherent mononuclear cells (Mo) were isolated from peripheral blood of healthy people and transfected with rAAV / CEA virus. The control group was stimulated with CEA polypeptide. Both groups were treated with recombinant human granulocyte macrophage colony stimulating factor GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-α (TNF-α) induced DC maturation. DCs were harvested on day 7, DCs were mixed with primary T cells, and cytotoxic T lymphocytes (CTLs) were induced. [3H] -TdR incorporation was used to detect the ability of DCs to proliferate autologous T cells. Flow cytometry was used to analyze the expression of IL- 4, the expression of IFN-γ, CD8, CD4, CD69 and CD25; take CEA-positive lung cancer cell line A549 as target cells, 51Cr release method was used to detect CTL killing efficiency of the target cells. Results The rAAV / CEA-transfected DC vaccine induced specific cellular immunity as follows: ⑧ The vaccine had a strong ability to stimulate lymphocyte proliferation, ⑧ CTL induced high expression of CD8, CD69 and IFN-γ, ⑧ CEA positive lung cancer target cells produce more killing activity, and this activity is antigen specific and MHC class I restriction. Conclusion AAV as a carrier, rAAV / CEA gene transfected DC can be successfully prepared for lung cancer therapeutic vaccine.