腺病毒介导HBx对人淋巴细胞影响的体外实验  

Effects of adenouvirus-mediated HBx on lymphocyte in vitro

作　　者：毕涛 卢倩倩 孙传东[3] BI Tao;LU Qianqian;SUN Chuandong(Department of Gastroenterology Surgery,Yantai Affiliated Hospital of Binzhou Medical University,Yantai 264100,Shandong,China;First Department of Oncology,Yantai Affiliated Hospital of Binzhou Medical University,Yantai 264100,Shandong,China;Department of Hepatobiliary Surgery,The Affiliated Hospital of Qingdao University,Qingdao 266000,Shandong,China)

机构地区：[1]滨州医学院烟台附属医院胃肠外科,山东烟台264100 [2]滨州医学院烟台附属医院肿瘤一科,山东烟台264100 [3]青岛大学附属医院肝胆外科,山东青岛266000

出　　处：《山东大学学报（医学版）》2018年第7期15-20,共6页Journal of Shandong University:Health Sciences

基　　金：山东省优秀中青年科学家科研奖励基金(2011GGH21842);山东省医药卫生科技发展计划(2012G0021845)

摘　　要：目的探讨体外条件下腺病毒介导乙型肝炎病毒x基因(HBx)在正常人外周血淋巴细胞中的复制扩增及其对淋巴细胞凋亡和白细胞介素-10(IL-10)分泌的影响。方法淋巴细胞取自正常人外周血,分别用携带HBx的腺病毒Ad-HBx、空载腺病毒Ad-EGFP感染淋巴细胞(设为Ad-HBx组和Ad-EGFP组),未感染腺病毒的淋巴细胞作为空白对照组(Lymphocyte组)。Q-PCR检测HBx在淋巴细胞内的复制扩增。ELISA检测3组淋巴细胞IL-10的表达。流式细胞术检测3组淋巴细胞的凋亡。结果 Q-PCR检测显示,Ad-HBx组淋巴细胞被Ad-HBx感染48 h后,细胞内HBx的表达较Ad-EGFP组和Lymphocyte组明显增高(P<0.05)。ELISA检测结果显示,Ad-HBx组淋巴细胞被Ad-HBx感染12、24、36、48 h后,IL-10的表达水平明显低于Ad-EGFP组和Lymphocyte组,差异有统计学意义(P<0.001)。流式细胞术检测结果显示,Ad-HBx组早期凋亡细胞明显多于其余2组,差异有统计学意义(P<0.05)。结论体外环境下,HBx在正常人外周血淋巴细胞内能够完成复制扩增,明显抑制淋巴细胞IL-10的表达和促进淋巴细胞的早期凋亡。HBx对人免疫系统的调控作用可能是通过在人淋巴细胞内的基因表达、抑制抗炎性因子表达和促进早期凋亡实现的。Objective To investigate the replication of adenovirus-mediated hepatitis B virus x gene（ Ad-HBx） in lymphocytes and its effects on the secretion of interleukin-10（ IL-10） and apoptosis of lymphocytes. Methods In vitro,lymphocytes collected from healthy human peripheral blood were transfected with Ad-HBx（ Ad-HBx group） and AdEGFP（ Ad-EGFP group）. Non-treated lymphocytes served as the blank controls（ Lymphocyte group）. The expression of HBx in lymphocytes was detected with quantitative polymerase chain reaction（ Q-PCR）. The secretion of IL-10 was assessed with enzyme linked immunosorbent assay（ ELISA）. The apoptosis of lymphocytes was detected with flow cytometry.Results Q-PCR results showed that the expression of HBx was significantly higher in the Ad-HBx group than in the Ad-EGFP group and Lymphocyte group（ P〈0.05） 48 h after transfection. ELISA results showed that the concentration of IL-10 was significantly lower in the Ad-HBx group than in the Ad-EGFP group and Lymphocyte group12 h,24 h,36 h and 48 h after transfection（ P〈0.001）. Flow cytometry results indicated that the apoptosis rate of Ad-HBxgroup in the early stage was significantly higher than that of the Ad-HBx group and Lymphocyte group（ P 〈0.05）.Conclusion In vitro,HBx is highly expressed in healthy human peripheral blood lymphocytes. Ad-HBx inhibits the expression of IL-10 and promotes lymphocyte apoptosis in an early stage. HBx affects the regulation of human immune system possibly through gene expression,promotion of early apoptosis and inhibition of anti-inflammatory cytokines.

关 键 词：腺病毒 乙型肝炎病毒X基因 淋巴细胞 白细胞介素-10 细胞凋亡 

分 类 号：R575[医药卫生—消化系统]