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-东南大学学报医学版

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

单位：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

分类号：Q813;R373.2

出版年·卷·期（页码）：2017·36·第五期（693-698）

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HepG2.2.15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（HepG2.2.15）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HepG2.2.15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、48h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HepG2.2.15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 flow 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 48h 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.

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