携带不同 HBV抗原基因片段的重组腺相关病毒转染树突状细胞诱导CTL的效应研究

目的：了解携带不同HBV抗原基因片段的重组腺相关病毒（rAAV‐HBV‐S、C、E、X）转染慢性乙型肝炎患者来源的树突状细胞（DC）诱导细胞毒性T淋巴细胞（CTL）的效应。方法采用携带不同HBV抗原基因片段的rAAV‐HBV‐S、C、E、X转染慢性乙型肝炎患者外周血中分离出的单核细胞，并在GM‐CSF、IL‐4和TNF‐α作用下继续培养7 d获得成熟的DC。通过观察DC的状态和流式细胞仪（FACS）检测各段 HBV转染后DC分化抗原（CD）的表达，评价其成熟与功能。将同一个体的DC与T细胞混合培养制备CTL ，通过四甲基偶氮唑盐（MTS）细胞杀伤实验研究被激活的CTL对HBV感染的靶细胞HepG2．2．15特异性的细胞毒作用。结果不同 HBV 抗原基因 rAAV‐HBV‐S、C、E、X 转染DC后 CD14、CD80、CD83、CD86的表型表达中， CD80、CD83差异有统计学意义（P＜0．05）；rAAV／HBV‐X转染的DC其CD80表达最高，rAAV／HBV‐S转染的DC其CD83表达最高。转染不同 HBV 抗原基因片段 rAAV（S、C、E、X）的 DC 诱导的 CTL 对 MHC‐Ⅰ类抗原阳性且有 HBV 的靶细胞（HepG2．2．15）的特异性杀伤效率显著高于对无 HBV的靶细胞（HepG2）的非特异性杀伤效应（P＜0．01），但不同rAAV‐HBV组间差异无统计学意义（P＞0．05）。结论 rAAV‐HBV‐S、C、E、X转染DC均可诱导CTL引起M HC‐Ⅰ依赖的特异性细胞毒效应。

Study on the killing effect of cytotoxic T lymphocytes induced by dendritic cells transduced recombinant adenovirus associated virus with different hepatitis B virus gene fragment

Objective To compare the killing effect of cytotoxic T lymphocytes(CTLs) induced by dendritic cells transduced recombinant adenovirus associated virus (rAAV ) with different hepatitis B virus gene fragment (rAAV‐HBV‐S ,C ,E ,X ) . Methods Peripheral blood mononuclear cells(PBMCs) from chronic hepatitis B patients were isolated and transduced recombinant adeno‐associated virus with different hepatitis B virus (HBV) antigen gene fragment (rAAV‐HBV‐S ,C ,E ,X) ,then GM‐CSF ,IL‐4 and TNFαwere added to cultivate for 7 days to generate mature dendritic cells (DCs) .The state of DCs were observed and differen‐tiation antigen molecules (CD)were detected by flow cytometry(FACS)to evaluate their maturation and function .Cytotoxic T lym‐phocytes (CTLs) were induced by the mixed cuture of DCs with prepared T lymphocytes .HepaG2 .2 .15 and HepaG2 were targed cells and the killing effect of CTLs wered compared using MTS assay .Results The expression of phenotype CD14 ,CD80 ,CD83 , CD86 from DCs transduced with rAAV‐HBV‐S ,C ,E ,X were compared ,respectively .Of which ,CD80 ,CD86 were significantly dif‐ferent(P<0 .05) .CD80 in group rAAV‐HBV‐X was the highest ,and CD83 in group rAAV‐HBV‐S was the highest .Cytotoxic effect of CTL induced by DC transduced with HBV antigen gene fragment of rAAV (S ,C ,E ,X) to target cells with HBV (HepG2 . 2 .15 cells) were significantly higher than that of without HBV target cells (HepG2 cells) (P<0 .01) ,but the four groups were no significant difference ,respectively(P>0 .05) .Conclusion HBV‐S ,C ,E ,X genes all induce M HC‐I dependently cytotoxic T‐lym‐phocytes specific response based adeno‐associated virus (AAV) vector delivery into dendritic cells .