肝癌特异性靶标致敏DC诱导CIK细胞对肝癌HuH-7细胞及移植瘤的抑制

Inhibiting effects of cytokine-induced killer cells co-cultured with hepatoma target peptide-sensitized dendritic cells on hepatoma cancer: Preliminary observations in vitro and in nude mice

目的:观察负载肝癌特异性DC靶标的树突状细胞(dendritic cell,DC)与细胞因子诱导的杀伤(cytokine-induced killer cell,CIK)细胞共培养后对肝癌细胞HuH-7的杀伤作用以及对裸鼠肝癌移植瘤的治疗效果。方法:外周血单个核细胞经不同细胞因子作用后培养成DC和CIK细胞。分别利用肝癌特异性DC靶标和肝癌HuH-7细胞冻融抗原刺激DC,ELISA法测定其对DC IL-12分泌的影响。DCHuH-7、DCtarget分别与CIK细胞共培养后,ELISA法检测其对CIK细胞IFN-γ分泌的影响,CCK-8法检测效靶比为10∶1、20∶1、40∶1、100∶1时DCHuH-7-CIK和DCtarget-CIK细胞对HuH-7细胞的杀伤作用。构建HuH-7细胞裸鼠皮下移植瘤模型,随机分为对照组、CIK组、DCHuH-7-CIK组和DCtarget-CIK组,尾静脉注射细胞悬液,观察效应细胞的抑瘤效果。结果:DCHuH-7与DCtarget的IL-12 p70分泌水平较DC显著升高[(179.33±14.04)、(173.33±6.66)vs(59.33±11.84)pg/ml,均P<0.01];与CIK细胞共培养后,DCHuH-7-CIK与DCtarget-CIK细胞分泌IFN-γ的能力也显著高于DC-CIK细胞(均P<0.01),且DCHuH-7-CIK与DCtarget-CIK细胞之间无显著差异(P>0.05)。在相同效靶比时,DCHuH-7-CIK细胞以及DCtarget-CIK细胞对HuH-7细胞的杀伤活性明显高于单纯CIK细胞(P<0.05),且DCHuH-7-CIK与DCtarget-CIK组之间无显著差异(P>0.05)。DCtarget-CIK细胞对裸鼠肝癌移植瘤的抑制作用与DCHuH-7-CIK细胞相仿,且均显著高于单纯CIK细胞[(78.48±14.58)%、(85.78±15.69)%vs(54.69±28.07)%,均P<0.05],无明显不良反应。结论:肝癌特异性DC靶标能够显著提高CIK细胞对HuH-7细胞及其裸鼠移植瘤的杀伤活性,可替代肝癌组织抗原负载DC。

Objective： To investigate the effects of cytokine-induced killer（ CIK） cells co-cultured with dendritic cells（ DCs） challenged with hepatoma target peptides on hepatoma cancer cell growth in vitro and in nude mice in vivo. Methods： Peripheral blood mononuclear cells（ PBMCs）,isolated from healthy volunteer subjects,were induced to differentiate into DCs and CIK cells with different cytokines,respectively. DCs were then stimulated with target peptides or whole cell lysates from human hepatoma HuH-7 cells to develop into DCHuH-7or DCtargetwhich were co-cultured with CIK cells for 6days to obtain DCHuH-7-CIK or DCtarget-CIK cells. Concentrations of IL-12 and INF-γ produced by CIK cells,DC-CIK cells,DCHuH-7-CIK or DCtarget-CIK cells were measured by ELISA. The cytotoxicity of these four types of effector cells against HuH-7 cells respectively was evaluated by CCK-8 assays in vitro and by HuH-7 cell transplantation assays in nude mice in vivo. Results： IL-12 production was significantly increased（ P 〈0. 01） in DCHuH-7-CIK cells（ 179. 33 ± 14. 04 pg /ml）and DCtarget-CIK cells（ 173. 33 ± 6. 66 pg /ml） as compared with DCs（ 59. 33 ± 11. 84 pg /ml）. Similarly,IFN-γ production was significantly increased in CIK cells co-cultured with DCHuH-7or DCtarget（ P 〈0.01） but was not significantly differ-ent between DCHuH-7-CIK and DCtarget-CIK cells（ P〉 0. 05）. Compared with untreated CIK cells,DCHuH-7-CIK cells and DCtarget-CIK cells possessed a similarly higher cytotoxic activity against HuH-7 cells in vitro（ P 〈0. 05） and a similarly stronger inhibitory effect on tumor growth in nude mice bearing hepatoma cancer（ [54. 69 ± 28. 07] vs [78. 48 ±14. 58]% and [85. 78 ± 15. 69]% respectively,P 〈0. 05）. Conclusion： Hepatoma target peptides-sensitized DCs may significantly enhance the cytotoxicity of CIK cells against human hepatoma cells,thus offering a potential strategy to enhance cell-based immunotherapy for liver cancer.