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目的研究肝素酶(Hpa)多肽疫苗体外能否诱发肝素酶特异性CTL反应,为肝素酶多肽疫苗的临床应用提供理论依据。方法5条HLA-A2限制性肝素酶抗原表位[Hpa(525-533)(PAFSYSFFV)、Hpa(353-361)(PLLSDTFAA)、Hpa(277-285)(KMLKSFLKA)、Hpa(400-408)(PLPDYWLSL)、Hpa(405-413)(WLSLLFKKL)]分别负载正常人外周血来源(PBMC)的树突状细胞(DC),诱导肝素酶特异性细胞毒T淋巴细胞(CTL),采用4 h标准51Cr释放实验检测上述CTL对不同肿瘤细胞的免疫杀伤效应,采用ELISPOT方法检测肝素酶表位肽负载的DC疫苗刺激的效应细胞IFN-γ的释放。结果5条肝素酶多肽表位中,只有Hpa(525-533)、Hpa(277-285)和Hpa(405-413)体外可以诱导肝素酶表位特异性CTL反应,对Hpa阳性且HLA-A2阳性的KATO-Ⅲ胃癌细胞、U2OS骨肉瘤细胞、SW480结肠癌细胞具有明显的杀伤效应,对HLA-A2阴性的HepG2肝癌细胞和Hpa阴性的MCF-7乳腺癌细胞不具有杀伤效应,但对转染了HLA-A2的HepG2细胞和转染了肝素酶质粒的MCF-7细胞恢复杀伤效应,对自体淋巴细胞不具有杀伤效应,进一步研究表明,Hpa(525-533)、Hpa(277-285)和Hpa(405-413)表位肽可以促进CTL细胞IFN-γ的释放。结论人肝素酶特异性抗原表位Hpa(525-533)、Hpa(277-285)、Hpa(405-413)不仅能激发机体特异性的抗肿瘤效应,而且还能诱导一个非特异性抗肿瘤的良性循环,这种肝素酶多肽疫苗具有广谱、高效、特异、安全的优点,为肝素酶表位多肽疫苗的临床应用提供了理论依据。
Objective To study whether heparanase (Hpa) vaccine can induce heparanase-specific CTL response in vitro and provide a theoretical basis for the clinical application of heparanase vaccine. Methods Five HLA-A2 restriction heparanase epitopes (Hpa (525-533) (PAFSYSFFV), Hpa (353-361) (PLLSDTFAA), Hpa (277-285) (KMLKSFLKA), Hpa (PLPDYWLSL) and Hpa (405-413) (WLSLLFKKL) were loaded with dendritic cells (DCs) derived from normal human peripheral blood (PBMCs) respectively to induce heparanase-specific cytotoxic T lymphocytes (CTLs) 4h standard 51Cr release test to detect the above CTL on different tumor cells immunosuppressive effect, ELISPOT method was used to detect heparin epitope peptide-loaded DC vaccine-stimulated effector cells IFN-γ release. Results Only five Hpa (525-533), Hpa (277-285) and Hpa (405-413) epitopes could induce heparanase epitope-specific CTL responses in vitro. Hpa (525-533), Hpa -A2-positive KATO-III gastric cancer cells, U2OS osteosarcoma cells and SW480 colon cancer cells have a significant killing effect, but have no killing effect on HLA-A2 negative HepG2 hepatoma cells and Hpa-negative MCF-7 breast cancer cells, HepG2 cells transfected with HLA-A2 and MCF-7 cells transfected with heparanase plasmid had no cytotoxic effect on autologous lymphocytes. Further studies showed that Hpa (525-533), Hpa (277 -285) and Hpa (405-413) epitope peptides can promote the release of IFN-γ from CTL cells. Conclusion Hpa (525-533), Hpa (277-285) and Hpa (405-413) can not only stimulate the body-specific anti-tumor effect, but also induce a nonspecific anti-tumor This heparinase polypeptide vaccine has the advantages of broad spectrum, high efficiency, specificity and safety, and provides a theoretical basis for the clinical application of the heparanase epitope peptide vaccine.