rAAV/HCV核心抗原基因转染树突状细胞的实验研究

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...

rAAV/hepatitis C virus core antigen gene tranfection into dendritic cell

Objective To study the feasibility of rAAV/hepatitis C virus(HCV) core antigen gene transfection into dendritic cell(DC).Methods The recombinant plasmid AAV(d16-95)/Corep5(rAAV/Core) was constructed and prepared by pSH3 packaging system.The mononuclear cells(DC precursor) from PMBC were isolated,infected by rAAV/Core virus stock,and induced by GM-CSF,IL-4 and TNF-.3 days later,the transfection efficiency(HCV core antigen positive ratio) was detected by flow cytometry,the HCV core antigen gene expression in DC was observed by fluorescence microscope.7 days later,the integration of rAAV/Core DNA and DC chromosome was detected by PCR/Southern blot analysis,the configuration of DC was observed by microscope,and the surface markers of DC including CD1a,CD40,CD80,CD83 and CD86 were measured by flow cytometry.Result rAAV/Core antigen gene was transfected into DC successfully,viral DNA was integrated into the DC chromosome,DC expressed the HCV core antigen gene and maintained typical morphologic feature with high expression of the surface markers.Conclusion The success of rAAV/Core antigen gene transfection into DC can make a base for HCV immunotherapy with DC.