南方医科大学学报 ›› 2014, Vol. 34 ›› Issue (04): 487-.

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rAAV/CEA转染树突状细胞制备肺癌疫苗

尤长宣,钱晓涛,何圆,Yong Liu,Paul L Hermonat   

  • 出版日期:2014-04-20 发布日期:2014-04-20

Development of a lung cancer vaccine by transfecting dendritic cells with rAAV/CEA

  • Online:2014-04-20 Published:2014-04-20

摘要: 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:①疫苗具备较强的
刺激淋巴细胞增殖的能力,②诱导的CTL较高表达CD8、CD69 和IFN-γ,③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。

Abstract: Objective To study the feasibility of preparing a therapeutic lung cancer vaccine by transfecting dendritic cells
(DCs) with adeno-associated virus vector carrying carcino-embryonic antigen gene (rAAV/CEA). Methods Adherent cells
(monocytes) isolated from the peripheral blood of a healthy donor were infected with rAAV/CEA virus stock or pulsed with
CEA peptide (control). The monocytes in both groups were induced into mature DCs with recombinant human GM-CSF, IL-4
and TNF-α. At day 7 of induction, the mature DCs were harvested and mixed with T lymphocytes. T cell proliferation
stimulated by the DCs was assessed with 3H-thymidine uptake, and the expression of IL-4, IFN-γ, CD8, CD4, CD25 and CD69
in cytotoxic T lymphocytes (CTL) was analyzed with flow cytometry. The cytotoxicity of the CTL against the target
CEA-positive lung cancer A549 cells was tested by 51Cr releasing assay. Results The DCs transfected with rAAV/CEA strongly
stimulated the proliferation of the T cell populations, and the induced CTL showed high expressions of CD8, CD69 and IFN-γ.
The transfected DCs exhibited a high killing ability of CEA-positive lung cancer cells, and the killing showed a CEA antigen
specificity and was limited by MHC I. These results suggested the ability of rAAV/CEA-transfected DCs in generating specific
cellular immunity in vitro. Conclusion It is feasible to prepare therapeutic lung cancer vaccines by transfecting DCs with rAAV/
CEA.