DC-CIK细胞对人肝癌HEPG2细胞周期、凋亡的影响

Effect of CIK cells combined with dendritic cells on cell cycle and apoptosis of human liver cancer cell line HEPG2

目的探讨细胞因子从肝癌患者外周血贴壁单个核细胞(peripheral blood mononuclear cells,PBMCs)诱导的树突状细胞(dendritic cells,DCs)与杀伤细胞(cytokine induced killer,CIK)对人肝癌HEPG2细胞系周期、凋亡的影响。方法用肝癌患者肝癌组织裂解物(肿瘤抗原)致敏经GM-CSF、TNF-α及IL-4等诱导该患者PBMC产生的DCs。用IFN-γ、IL-2和人CD3单克隆抗体(human CD3 monoclonal antibody,hCD3mAb)等诱导该PBMC的悬浮细胞产生CIK细胞。用Transwell培养小室将DC-CIK细胞与HEPG2细胞在同一培养体系内经该小室膜分隔培养24、48 h。流式细胞仪检测该HEPG2细胞的周期;Annexin V-PI双染法检测其凋亡。RT-PCR、Western blot分别检测凋亡相关基因Bax及其编码蛋白的表达。结果 DC-CIK细胞可明显影响人HEPG2细胞生长周期,两者共培养,可将HEPG2细胞系阻滞于G1期。DC-CIK细胞能显著诱导HEPG2细胞的凋亡,与对照相比,其凋亡诱导率差异显著(P<0.01)。基因与蛋白水平检测表明,DC-CIK细胞可致HEPG2细胞系的凋亡相关基因Bax表达明显上调。结论肝癌患者DC-CIK细胞可明显抑制肝癌HEPG2细胞系的生长,显著诱导该细胞凋亡,且该凋亡诱导具"时间-依赖"与"细胞数量-依赖"特性。

Objective To explore the effect of cytokine induced killer（CIK） cells and dendritic cells（DCs） from peripheral blood mononuclear cells（PBMC） of patients with liver cancer on cell cycle and apoptosis of human liver cancer cell line HEPG2.Methods Adherent PBMCs were induced by GM-CSF,TNF-α and IL-4 to prepare DCs,which were sensitized with antigen of autologous tissue of liver cancer.CIK cells were obtained from suspended PBMCs induced by IFN-γ,IL-2 and human CD3 monoclonal antibody（hCD3mAb）.DCs-CIK cells and HEPG2 cell line were cultured in a transwell chamber,and were cultured separately by the membrane of transwell chamber for 24 h and 48 h,respectively.Cell cycle of HEPG2 cell line that was cultured with DCs-CIK cells for 24 h and 48 h was detected by flow cytometry（FCM）.Annexin V-PI staining was applied for analysis of HEPG2 cell line apoptosis.mRNA and protein expression of Bax was determined by reverse transcriptase-polymerase chain reaction（RT-PCR） and Western blotting,respectively.HEPG2 cell line that was cultured without DCs-CIK cells was set as control.Results DCs-CIK cells markedly affected the cell cycle of HEPG2 cell line,which was blocked at G1 stage when they were cultured together.DCs-CIK cells significantly induced HEPG2 cell line apoptosis compared with the control（P〈0.01）.DCs-CIK cells significantly up-regulated mRNA and protein expression of Bax in HEPG2 cell line.Conclusion DCs-CIK cells of patient with liver cancer can apparently inhibit the growth of human cancer cell line HEPG2,and can induce cell apoptosis in a time-dependent and cell population-dependent manner.