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目的:研究rAAV/丙型肝炎病毒核心抗原基因[hepatitis C virus(HCV)core gene]转染树突状细胞(dendritic cell,DC)的可行性。方法:构建重组质粒AAV(d16-95)/Core p5(简称rAAV/Core),并以pSH3包装系统制备该病毒。分离外周血单个核细胞(DC前体细胞),以rAAV/Core病毒转染DC前体细胞(基因转染组),采用巨噬细胞集落刺激因子、白介素-4、肿瘤坏死因子-α诱导成熟。3d后,收集部分DC,以流式细胞仪检测rAAV/Core转染效率及HCV核心抗原在DC中的表达情况。7d后,收集细胞,PCR/Southern blot分析rAAV/Core DNA在DC染色体的整合情况。显微镜下观察DC形态,流式细胞仪检测DC表面分子标志CD1a、CD40、CD80、CD83、CD86的表达情况。结果:rAAV/Core成功转染DC,质粒DNA整合到DC染色体上,HCV核心抗原基因在DC内表达,DC成熟不受病毒转染影响,具有典型DC外观并高表达特异的成熟DC表面标志。结论:rAAV/Core转染和制备DC的成功为丙型病毒性肝炎的DC免疫治疗打下实验室基础。
Objective: To investigate the feasibility of transfecting dendritic cells (rAAV / HCV core gene) with dendritic cells (DCs). Methods: The recombinant plasmid AAV (d16-95) / Core p5 (rAAV / Core) was constructed and the virus was prepared by the pSH3 packaging system. Peripheral blood mononuclear cells (DC precursor cells) were isolated, DC precursor cells (gene transfection group) were transfected with rAAV / Core virus, and induced by macrophage colony stimulating factor, interleukin-4 and tumor necrosis factor-α . After 3 days, DCs were collected and the rAAV / Core transfection efficiency and the expression of HCV core antigen in DC were detected by flow cytometry. After 7 days, the cells were harvested and the integration of rAAV / Core DNA in the DCs was analyzed by PCR / Southern blot. The morphological changes of DCs were observed under a microscope. The expression of CD1a, CD40, CD80, CD83 and CD86 were detected by flow cytometry. RESULTS: DCs were successfully transfected with rAAV / Core, and the plasmid DNA was integrated into the DC chromosome. HCV core antigen gene was expressed in DCs. DC maturation was unaffected by virus transfection, with typical DC appearance and high expression of specific mature DC surface markers. CONCLUSIONS: The success of rAAV / Core transfection and preparation of DCs provided a laboratory basis for DC immunotherapy with hepatitis C virus.