【作者】 章欢；

【导师】 张伦理；

【作者基本信息】 南昌大学 ， 内科学， 2010， 硕士

【摘要】 目的:构建表达乙肝病毒核心抗原(HBcAg)的复制缺陷型重组腺病毒载体,将其分别转染健康人及乙肝病毒携带者外周血单个核细胞,观察其刺激产生α干扰素水平并与TLR9的天然配体CpG ODN 2006刺激效果进行比较,以初步探讨乙型肝炎病毒感染免疫识别状态及乙肝病毒感染慢性化机制。方法:扩增乙肝病毒(HBV)C基因片段,亚克隆到穿梭质粒pAdTrack-CMV上,与5型腺病毒骨架质粒pAdeasy-1共同电转化大肠杆菌BJ5183内进行同源重组,经卡那霉素抗性筛选和酶切鉴定筛选出携带(HBV)C区基因的重组腺病毒载体,用脂质体包裹PacⅠ酶切线性化的重组质粒,转染到293T细胞内进行重组腺病毒的包装,获得具有感染能力的重组腺病毒颗Ad-HBc。应用密度梯度离心法从健康人(HBsAg阴性)及乙肝病毒携带者外周静脉血中分离出单个核细胞,分为四组加入干预因素:Ad-HBc组、CpG ODN 2006组、Ad-HBc+CpG ODN 2006组和空白对照组,孵育48小时后采用ELISA检测各组细胞培养上清中IFN-α水平。结果:在健康人中,与空白对照组比较,Ad-HBc+CpG ODN 2006组、Ad-HBc组和CpG ODN 2006组中IFN-α均有明显升高(P<0.05),Ad-HBc+CpG ODN 2006组升高最明显,Ad-HBc组次之,CpG ODN 2006组最低。在携带者中,与空白对照组比较,Ad-HBc+CpG ODN 2006组升高较明显,CpG ODN 2006组次之,Ad-HBc组最低(P<0.05),且Ad-HBc组水平明显低于健康人同组水平(P<0.05)。结论:携带乙肝核心抗原基因复制缺陷型重组腺病毒感染健康人外周血单个核细胞能诱导增加健康人PBMC产生IFN-α的能力,且刺激效果优于TLR9的配体CpG ODN 2006。而在乙肝病毒携带者中,重组腺病毒感染外周血单个核细胞产生IFN-α水平低于健康人,提示与HBV持续感染造成外周血pDC功能下降有关,为进一步研究HBV持续感染的免疫机制提供了思路。更多还原

【Abstract】 Objective:To construct recombinant adenoviral vector carrying HBcAg gene by homologous recombination in bacteria and to transfect human peripheral blood mononuclear cells from healthy subjects and HBV carriers, analyze the value of IFN-αin culture supernatants by ELISA and compare to the results of CpG ODN 2006 (ligand of TLR9) in purpose of investigating the mechanism of hepatitis B virus infection recognition in immune system.Methods:HBV C genes were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV. Then the resultant pAdTrack-CMV-HBc was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBV C gene (pAd-HBc) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 T cells. The recombinant adenoviruses Ad- HBc were obtained. With the method of density gradient centrifugation,isolating peripheral blood mononuclear cells from healthy subjects(HBsAg negative) and HBV carriers were divided into four groups. Three groups was stimulated respectively by Ad-HBc+CpG ODN 2006、Ad-HBc、CpG ODN 2006 and the fourth was control group. After 48 hours, the value of IFN-αin culture supernatants was detected by ELISA.Results:In healthy subjects, compared with control, the concentration of IFN-αin Ad-HBc+CpG ODN 2006 group、Ad-HBc group and CpG ODN 2006 group were all elevated(P<0.05),Ad-HBc+CpG ODN 2006 group increased significantly,Ad-HBc group followed,CpG ODN 2006 group the lowest. In HBV carriers, compared with control, Ad-HBc + CpG ODN 2006 group increased slightly ,CpG ODN 2006 group followed, Ad-HBc group the lowest (P<0.05).The IFN-αconcentration of Ad-HBc in HBV carriers is significantly lower than healthy subjects(P<0.05).Conclusions:Replication- deficient recombinant adenoviral vector carrying HBcAg gene infected human peripheral blood mononuclear cells from healthy subjects, increased ability of pDC can be induced to produce IFN-α.The stimulated result was more effective than the ligand CpG ODN 2006 of TLR9. In HBV carriers, the adenovirus infection of peripheral blood mononuclear cells were disable to produce IFN-αmore than CpG ODN 2006,the result may involve in dysfunction of pDCs caused by persistent infection of HBV.更多还原