带有HBV基因的腺相关病毒为载体的重组病毒感染树突状细胞的研究  

Transduction of dendritic cells by recombinant adeno-associated virus （rAAV） carrying hepatitis B virus antigens

作　　者：尤红[1] 丛敏[1] 王萍[1] 阎钟钰[1] 徐雍[1] 卢炎[1] 王宝恩[1] 贾继东[1] 

机构地区：[1]首都医科大学北京友谊医院肝病中心,100050

出　　处：《中华实验和临床病毒学杂志》2007年第2期105-107,共3页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的探讨腺相关病毒（Adeno-associated virus，AAV）为载体的含有HBV—S、C或X基因的重组病毒（rAAV—HBV—S，C，X）感染正常人树突状细胞的效率。方法分别构建含有HBV-S、C和x基因的从v质粒，在293细胞中包装成重组病毒，测定病毒滴度。从正常人外周血中分离出单核细胞进行体外培养，在培养的第1天分别用rAAV—HBV—S、C、X和作为对照的293细胞裂解液感染刺激单核细胞，感染后的单核细胞在GM-CSF、IL-4和TNF-α作用下继续培养7d获得成熟的树突状细胞。用RT-PCR及流式细胞仪细胞内染色检测HBV基因的RNA和蛋白的表达。结果重组rAAV—HBV—S、C、X的病毒滴度可以达到10^7拷贝／ml，用重组病毒分别感染树突状细胞。与对照组相比，感染的树突状细胞可表达相应的HBV基因RNA，流式细胞仪检测显示约90％的树突状细胞有相应HBV蛋白的表达。结论rAAV-HBV可有效感染树突状细胞，提示可以通过该途径刺激树突状细胞提呈抗原。Objective To test the efficiency of infection of recombinant adeno-associated virus （rAAV） carrying hepatitis B virus S, C or X antigen （rAAV-HBV-S, C, X）to human dendritic ceils. Methods Recombinant AAV plasmids containing HBV-S, C or X gene were constructed and packaged into rAAV in 293 cells. Monocytes were isolated from healthy donor and pulsed by rAAV-HBV-S, X, C or 293 lysate as control at the first day of isolation, then the dentritic cells were cultured for 7 days in vitro. The transcription and expression of HBV-S, C or X gene were analyzed by reverse transcription-polymerase chain reaction （RT-PCR） or intracellular staining with fluorescence activated cell sorter （ FACS）, respectively. Results The titer of rAAV-HBV-S, C, X virus was approximately 10^7 copies per ml. After infection the HBV-S, C or X transcription expression could be seen by RT-PCR in the infected dendritic cells, the efficiency was about 90 percent by FACS. Conclusion rAAV-HBV-c can effectively infect and pulse dendritic cells.

关 键 词：依赖病毒 肝炎病毒 乙型 树突细胞 

分 类 号：R686[医药卫生—骨科学]