肝素酶基因修饰的树突状细胞疫苗对胃癌细胞的免疫效应研究

目的研究肝素酶基因修饰的树突状细胞(DC)疫苗对胃癌细胞的免疫效应,探讨肝素酶疫苗在胃癌主动免疫治疗中的可行性。方法将含有肝素酶全长cDNA的重组肝素酶腺病毒感染人类白细胞抗原A2(HLA-A2)阳性健康志愿者外周血单个核细胞来源的DC,制备肝素酶基因修饰的DC疫苗,刺激同一来源的淋巴细胞,使其活化为肝素酶特异性细胞毒T淋巴细胞(CTL),采用标准51Cr释放试验验证其对KATO-Ⅲ和SGC-7901胃癌细胞的体外免疫效应,肝素酶特异性的CTL与不同靶细胞共孵育后干扰素(IFN)-γ的释放采用酶联免疫吸附试验(ELISA)法。结果经肝素酶重组腺病毒感染后该DC中肝素酶蛋白质的表达明显升高。肝素酶特异性的CTL在各效/靶比时对HLA-A2及肝素酶均阳性的KATO-Ⅲ胃癌细胞都有明显的免疫杀伤活性;相反,对肝素酶阳性但HLA-A2阴性的SGC-7901胃癌细胞不具有杀伤效应,即使在最高效/靶比时,其杀伤活性亦仅为11·1%±4·6%;进一步研究发现肝素酶特异性CTL对自体淋巴细胞亦不具有免疫杀伤作用,在最高效/靶比时,其杀伤活性为11·4%±7·9%。同时研究还发现,肝素酶修饰的DC刺激的效应细胞与KATO-Ⅲ共孵育后,其IFN-γ的表达明显高于空病毒修饰组以及IL-2刺激组(280pg/ml±24pg/mlvs121pg/ml±19pg/ml和60pg/ml±11pg/ml,P<0·05);而经肝素酶修饰的DC刺激的特异性效应细胞与SGC-7901或自体淋巴细胞孵育后所检测的IFN-γ在各组间差异无统计学意义(均P>0·05)。结论肝素酶基因修饰的DC疫苗可以诱发特异性CTL,对HLA相匹配且肝素酶阳性的胃癌细胞具有良好的免疫杀伤活性,而对自体淋巴细胞不具有免疫杀伤作用,是一种安全有效的肿瘤免疫基因治疗的靶位。

Immune response of heparinase gene modified dendritic cell-based vaccine on gastric cancer cells

OBJECTIVE: To explore the feasibility of heparinase vaccine in active immunity for gastric cancer. METHODS: Dendritic cells originated from the peripheral blood mononuclear cells (PBMC) of healthy HLA-A2 positive donors were transfected with recombinant adenovirus containing heparinase full length cDNA of heparanase to generate heparanase gene modified DC vaccine. T lymphocytes from the same donors were activated by those genetically modified DC vaccine repeatedly to generate heparanase specific cytotoxicity T lymphocytes (CTL). CTL-mediated cell lysis to gastric cancer cells of the lines KATO-III and SGC-7901 was analyzed in vitro by standard (51)Cr releasing assay. Heparinase specific CTL were co-cultured with KATO-III and SGC-7901 cells, and then ELISA was used to detect the IFN-gamma release. RESULTS: Expression of heparanase was significantly increased in the DCs transfected with heparinase recombinant adenovirus. Heparanase specific CTL generated from the genetically modified DC vaccine exhibited potent lysis to the KATO-III gastric cancer cells positive in both heparinase and HLA-A2 at each E/T ratio, whereas, these heparinase specific CTL could not lyse the SGC-7901 cells positive to heparinase but negative to HLA-A2, with a specific lysis rate of only 11.1% +/- 4.6% even at an E:T ratio of 40:1. Further study showed that heparanase vaccination had no detectable lysis on the autologous lymphocytes in vitro with a specific lysis rate of only 11.4% +/- 7.9% even at an E:T ratio of 40:1. The IFN-gamma release amount when the heparanase specific CTL were co-cultured with the KATO-III cells was 280.4 pg/ml +/- 23.5 pg/ml, significantly higher than that when the heparanase specific CTL were co-cultured with the rAd5-Lacz modified DC (120.6 pg/ml +/- 18.9 pg/ml), and that of the IL-2 stimulated T cells (60.0 pg/ml +/- 10.6 pg/ml, both P < 0.05). In contrast the IFN-gamma release amounts of the SGC-7901 cells and autologous lymphocytes remained unchanged when they were co-cultured with either above-mentioned effector cells (both P > 0.05). CONCLUSION: DC genetically modified by heparanase gene activate heparanase specific CTL and induce potent immune response against HLA-matched and heparinase positive gastric cancer cells in vitro, whereas they have no killing effect on autologous lymphocytes. Heparanase is an effective and safe target for immunogen therapy of tumor, thus providing a new biotherapy method for advanced gastric cancer.