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目的探讨经细胞因子从肝癌患者外周血贴壁单个核细胞(PBMCs)诱导的树突状细胞(DC)与杀伤细胞(CIK)对人肝癌HepG2细胞增殖、迁移的影响。方法用肝癌患者肝癌组织裂解物(肿瘤抗原)致敏经粒细胞巨噬细胞刺激因子(GM-CSF)、肿瘤坏死因子-α(TNF-α)及白细胞介素-4(IL-4)等诱导该患者PBMCs产生的DC。用干扰素-γ(IFN-γ)、IL-2和人CD3单克隆抗体(human CD3monoclonal antibody,hCD3mAb)等诱导该PBMCs的悬浮细胞产生CIK细胞。用Tanswell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、48h。CCK-8法检测HepG2细胞生长变化,并绘制其生长曲线;划痕实验检测其增殖、迁移能力。RT-PCR、Western blot分别检测其增殖细胞核抗原(PCNA)基因及其编码蛋白的表达。结果 CCK-8法检测显示,DC-CIK细胞对人HepG2细胞生长具显著杀伤作用,与对照组细胞比较,差异有统计学意义(P<0.01)。划痕实验、基因与蛋白水平检测表明,DC-CIK细胞可明显抑制该瘤细胞的增殖与迁移,能在基因与蛋白水平下调该瘤细胞PCNA的表达。结论 DC-CIK细胞可显著下调肝癌细胞的PCNA基因表达,明显抑制其增殖与迁移,以发挥其杀瘤作用。
Objective To investigate the effects of dendritic cells (DCs) and killer cells (CIKs) induced by peripheral blood mononuclear cells (PBMCs) on the proliferation and migration of HepG2 cells induced by cytokines. Methods GM-CSF, TNF-α, IL-4, etc. were sensitized by liver cancer tissue lysate (tumor antigen) DCs induced by this patient’s PBMCs were induced. CIK cells were induced by suspension cells of this PBMCs with interferon-γ (IFN-γ), IL-2 and human CD3 monoclonal antibody (hCD3 mAb). DC-CIK cells were cultured with HepG2 cells in Tanswell culture chamber for 24,48 h in the same culture system. CCK-8 method was used to detect the growth of HepG2 cells and the growth curve was drawn. Scratch assay was used to detect the proliferation and migration ability of HepG2 cells. RT-PCR and Western blot were used to detect the expression of proliferating cell nuclear antigen (PCNA) gene and its encoded protein. Results The CCK-8 assay showed that DC-CIK cells had a significant killing effect on human HepG2 cells. Compared with the control cells, the difference was statistically significant (P <0.01). Scratch experiments, gene and protein levels showed that DC-CIK cells can significantly inhibit the proliferation and migration of the tumor cells, and can down-regulate the expression of PCNA at the gene and protein levels. Conclusion DC-CIK cells can significantly down-regulate the expression of PCNA gene in hepatocellular carcinoma cells and significantly inhibit the proliferation and migration of hepatocellular carcinoma cells in order to exert its antitumor effect.