论文部分内容阅读
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。
Objective To study the immune effect of heparanase gene modified dendritic cell (DC) vaccine on gastric cancer cells and to explore the feasibility of heparinase vaccine in active immunotherapy of gastric cancer. Methods DCs derived from peripheral blood mononuclear cells of human leukocyte antigen A2 (HLA-A2) positive healthy volunteers were infected with recombinant heparinase adenovirus containing heparanase full-length cDNA to prepare DC vaccine modified by heparanase gene and stimulated Lymphocytes from the same source were activated to heparanase-specific cytotoxic T lymphocytes (CTLs). The standard 51Cr release assay was used to verify the in vitro immune response to KATO-III and SGC-7901 gastric cancer cells. Heparinase specificity The release of interferon (IFN) -γ was detected by enzyme-linked immunosorbent assay (ELISA) after co-incubation of CTL with different target cells. Results The heparinase protein expression in DCs was significantly increased after heparinase recombinant adenovirus infection. Heparinase-specific CTLs exhibited significant immunosuppressive activity against KATO-III gastric cancer cells both HLA-A2 and heparanase at each effect / target ratio; on the contrary, hematinase-positive but HLA-A2-negative Of SGC-7901 gastric cancer cells did not have killing effect, even in the most efficient / target ratio, its killing activity was only 11.1% ± 4.6%; further study found that specific CTL heparin on autologous lymphocytes also It has no immunogenicity and its killing activity is 11.4% ± 7.9% at the highest efficiency / target ratio. At the same time, it was also found that the expression of IFN-γin heparin-modified DC stimulated cells co-incubated with KATO-Ⅲ was significantly higher than that in empty virus modified group and IL-2stimulated group (280pg / ml ± 24pg / ml vs121pg / ml ± 19 pg / ml and 60 pg / ml ± 11 pg / ml, P <0.05); while the specific effector cells stimulated with heparanase-modified DCs incubated with SGC-7901 or autologous lymphocytes There was no significant difference of -γ between groups (all P> 0.05). Conclusion The DC vaccine modified by heparanase gene can induce specific CTL, which is safe and effective for HLA-matched and heparanase-positive gastric cancer cells with good immunosuppressive activity but not immune lymphocyte autologous lymphocytes Of the tumor immune gene therapy target.