肝癌相关抗原Kinectin蛋白致敏树突状细胞诱导的CTL对肝癌细胞株杀伤作用的检测

CTL induced by kinectin-MBP-impulsed dendritic cells specifically kills kinectin positive human hepatocellular caner cells

目的探讨肝癌相关抗原Kinectin基因重组蛋白Kinectin-MBP(MBP,麦芽糖结合蛋白)致敏树突状细胞(DCs)体外诱导特异性细胞毒T淋巴细胞(CTL)对肝癌细胞株的杀伤作用。方法体外基因工程的方法表达、纯化Kinectin-MBP。从正常人外周血中分离单个核细胞(PBMC),重组人粒细胞-巨噬细胞集落因子(rhGM-CSF)、重组人白细胞介素4(rhIL-4)联合培养,诱导扩增DCs,并通过形态学、流式细胞术鉴定DCs。DCs经Kinectin-MBP致敏后,ELISA检测细胞上清液中IL-12和IFN-γ的含量;将致敏DCs与自体T淋巴细胞混合培养后,MTT法检测T细胞增殖的能力;同时诱导T细胞增殖转化为CTL,以此CTL为效应细胞,Kinectin表达阳性的肝癌细胞株bel-7404为靶细胞,LDH 4 h释放法检测CTL对肝癌细胞株的杀伤作用。结果体外重组蛋白技术成功表达、纯化出Kinectin-MBP。PBMC经rhGM-CSF和rhIL-4联合诱导,可成功培养出DCs。DCs经Kinectin-MBP致敏后上清液中IL-12和IFN-γ的分泌量明显增高,且刺激自体T细胞增殖的能力也显著增强;同时Kinectin-MBP致敏的DCs能明显诱导CTL的增殖,此CTL对Kinectin阳性的7404肝癌细胞株有较明显的杀伤作用,其杀伤率在效靶比为25∶1时达最高峰[为(65.00±1.47)%],显著高于其他对照组(均P<0.001)。结论体外诱导扩增的DCs经肝癌相关抗原Kinectin-MBP致敏后具有较强的刺激自体T细胞增殖的能力,且在体外可诱导同种CTL产生和增殖,后者对Kinectin阳性的7404肝癌细胞株具有较强的杀伤效应。该实验为将Kinectin蛋白运用于以DCs为基础的肝癌临床免疫治疗奠定了基础。

【Objective】 To study the anti-tumor effect of cytotoxic T lymphocyte（CTL）,which was induced by recombinant protein kinectin-MBP-impulsed dendritic cells（DCs）,against human hepatocellular carcinoma（HCC）.【Methods】 Kinectin-MBP fusion protein were expressed and purified by means of recombinant protein technology in vitro,DCs were induced with rhGM-CSF and IL-4 from human peripheral bloodmononuclear cells（PBMCs）.DCs impulsed with kinectin-MBP were co-cultured with T lymphocytes,and the resultant CTLs were used as effector cells,kinectin positive hepatocellular carcinoma bel-7404 cells were used as target cells to test the specific antitumor effect of CTL using LDH 4 hours release.【Results】 Successfully expressed and purified kinectin-MBP by used of recombinant protein technology in vitro.After induced by rhGM-CSF and IL-4,PBMCs presented typical morphologic characteristics of DC.The level of CIL-12 and CIFN-γ raised significantly in culture medium supernatants of kinectin-MBP treated group;The proliferation capacity of autologous T cell cultured with kinectin-MBP-pulsed DC were significantly higher than MBP-pulsed,non-treated cultured DC group（P〈0.05）.Higher anti-tumor activity was found by DC-induced the CTL of the BEL-7404 hepatoma cells in kinectin-MBP-pulsed groups.The highest killing rate was found when the ratio of effector：target was 25：1（65.00±1.47%）.【Conclusion】 The DCs sensitized by Kinectin-MBP can obtain the capability of stimulating autologous T cells＇ proliferation.The pulsed DC can also induce CTL successfully,and have strong cytotoxic activity toward the BEL-7404.Kinectin protein will be the foundation of DC-based immunotherapy of clinical liver cancer.