DC-CIK与CIK体外培养对包含HBV基因的HepAD38细胞的杀伤效果研究

Study the Killing Effect of DC-CIK and CIK in Vitro on HepAD38 Cells Containing HBV Gene

目的:研究乙型肝炎病毒抗原致敏的树突状细胞(DC)和CIK细胞共同培养后对Hep AD38细胞(含整合HBV基因组)的杀伤作用。方法:从乙肝患者外周血中提取外周血单个核细胞(peripheral blood mononuclear cell,PBMC),平均分为两组,研究组培养DC,培养DC第5天加入乙肝抗原并收获DC,然后加入CIK细胞,培养出DC-CIK细胞;对照组单独培养CIK细胞。两组在培养15 d后分别收获DC-CIK细胞与CIK细胞,同时作为效应细胞,把含有乙肝病毒的Hep AD38细胞作为靶细胞,分别在5∶1、10∶1的效靶比时进行杀伤试验,使用LDH释放法测定杀伤活性,并用荧光扩增法检测杀伤试验后培养基内HBV-DNA水平。结果:研究组的DC-CIK细胞的5∶1效靶比时的平均杀伤率为(54.4±6.3)%、10∶1效靶比时的平均杀伤率为(71.5±4.5)%,均明显高于对照组CIK细胞的(42.4±3.0)%、(59.4±4.5)%,差异均有统计学意义(P<0.05)。研究组的DC-CIK细胞5∶1效靶比时的培养基内的HBV-DNA平均为(1.20±0.30)×106/m L、10:1效靶比时的培养基内的HBV-DNA平均为(1.64±0.60)×107/m L,均明显高于对照组CIK细胞的(0.90±0.23)×106/m L、(1.28±0.23)×107/m L,差异均有统计学意义(P<0.05)。结论:CIK细胞与DC-CIK细胞对Hep AD38细胞均具有杀伤效果,而且随着效靶比的升高,杀伤效果增强。同时,乙肝抗原致敏的DC诱导出的特异性CIK细胞(DC-CIK细胞),能够有效提高CIK细胞对乙肝病毒感染的Hep AD38细胞的杀伤作用,其杀伤作用可能与DC被乙肝抗原致敏后诱导出特异性CIK,增强各自疗效有关。

Objective：To study the killing effect of hepatitis B virus antigen sensitized dendritic cells（DC） and CIK cells on Hep AD38 cells（including the integrated HBV genome） after co culture.Method：PBMC was extracted from peripheral blood of hepatitis B patients and divided into two groups,the study group cultured DC,cultured DC for fifth days,added hepatitis B antigen and harvested DC,then added CIK cells to culture DC-CIK cells;the CIK cells were cultured alone in the control group.In the two groups,DC-CIK cells and CIK cells were harvested after 15 d culture,and as the effector cells,the Hep AD38 cells containing HBV were used as target cells,in effect the target 5∶1,10∶1 ratio was measured using LDH cytotoxicity assay,cytotoxicity release assay and fluorescence amplification method to detect cytotoxicity after HBV-DNA in the culture medium.Result：The mean killing rate of DC-CIK cells at 5：1,10：1 target ratio in study group were（54.4±6.3）% and（71.5±4.5）%,higher than（42.4±3.0）% and（59.4±4.5）% of control group,the differences were statistically significant（P〈0.05）.The level of HBV-DNA culture medium of 5∶1 cells and 10∶1 cells in the target ratio of DC-CIK in study group were（1.20±0.30）×10^6/m L and（1.64±0.60）×10^7/m L,higher than（0.90±0.23）×10^6/m L and（1.28±0.23）×10^7/m L of control group,the differences were statistically significant（P〈0.05）.Conclusion：CIK cells and DC-CIK cells have a lethal effect on Hep AD38 cells,and the effect target ratio increased,the killing effect is enhanced.At the same time,the hepatitis B antigen sensitized DC induced specific CIK cells（DC-CIK cells）,can effectively improve the killing effect of CIK cells on hepatitis B virus infection of Hep AD38 cells and its cytotoxicity DC and hepatitis B antigen sensitization induced specific CIK,enhance their curative effect.