共刺激基因工程细胞共培养的细胞因子诱导的杀伤细胞对HepG2.2.15细胞中乙肝病毒的抑制作用研究  

Study on the inhibitory effect of ECCE-CIK on the HBV replication in HepG2.2.15 cells

作　　者：蒋文秀[1] 王利利[1] 叶伟[1] 范璟[1] 殷丹丹[1] 张亚娟[2] 赵伟[1] 

机构地区：[1]东南大学附属第二医院,江苏南京210003 [2]丹阳市人民医院,江苏镇江212300

出　　处：《东南大学学报（医学版）》2017年第5期693-698,共6页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(81402559)

摘　　要：目的:研究共刺激基因工程细胞共培养的细胞因子诱导的杀伤细胞(ECCE-CIK)对人肝癌细胞系Hep G2.2.15细胞中乙肝病毒的抑制作用。方法:将共刺激基因工程细胞与人外周血单个核细胞共同培养,使用IFN-γ、CD3单抗、IL-2细胞因子诱导,产生ECCE-CIK细胞;将效应细胞(ECCE-CIK)和靶细胞(Hep G2.2.15)以效∶靶1∶1、5∶1、20∶1的比例共同孵育,使用CFSE/PI染色法检测ECCE-CIK对Hep G2.2.15的杀伤作用;建立体外直接法与间接法共培养系统,收集作用后3、24、48 h的培养上清液,检测HBV DNA与HBsAg水平。结果:随着效靶比的增加和时间的延长,杀伤率逐渐上升,培养上清中HBV DNA、HBsAg水平逐渐下降(P<0.05);直接系统对HBV DNA、HBsAg的抑制曲线略高于间接系统。结论:在体外直接法与间接法共培养系统中,ECCE-CIK都能降低Hep G2.2.15中HBV DNA和HBsAg水平,提示ECCE-CIK能通过细胞毒和非细胞毒方式抑制靶细胞中乙肝病毒的复制,为临床治疗提供了实验依据。Objective： To study the effect of the Cytokine-induced killer cells cultured with engineered cells for costimulatory enhancement（ ECCE-CIK） on inhibiting the replication of HBV produced by the human hepatoma cell lines HepG2.2.15.Methods： To generate ECCE-CIKs,ECCE were co-cultured with the peripheral blood mononuclear cells,IFN-γ,anti-CD3 antibody,and IL-2 were added into the culture system later.Then effector cells（ ECCE-CIK） were co-cultured with target cells（ HepG2.2.15） at effector to target（ E∶ T） ratios of 1∶ 1,5∶ 1,20∶ 1.After that the method of CFSE/PI was used to evaluate the death of target cells by ow cytometry.Then direct culturing system and indirect culturing system were established for the co-culturing of effector cells and target cells in vitro.At last the supernatants were collected from the two systems at 3,24 and 48 h respectively for detecting the amount of HBV DNA and HBsAg.Results： Accompanied by the increasement of E∶ T ratios and the duration of co-culturing the percentage of CFSE^＋PI^＋cells were increased progressively,while the levels of HBV DNA and HBsAg were decreased（ P〈0.05）.Conclusion： These results suggestes that HBV replication in HepG2.2.15 can be suppressed by ECCE-CIK in both direct system and indirect system through cytolytic and noncytolytic mechanisms which may have a good clinical prospect.

关 键 词：细胞因子诱导的杀伤细胞 共刺激基因工程细胞 细胞毒作用 非细胞毒作用 乙肝病毒 

分 类 号：Q813[生物学—生物工程] R373.2[医药卫生—病原生物学]