光动力法制备DC胶质瘤疫苗抗肿瘤作用的研究

Antitumor efficacy of photodynamic therapy-generated glioma vaccine from dendritic cells

目的:研究光动力法(photodynamic therapy,PDT)处理后,用酸洗法获取C6胶质瘤细胞的抗原肽,制备树突状细胞(dendritic cell,DC)疫苗,诱导细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)在体外杀伤C6细胞的活性,以及PDT对C6胶质瘤细胞免疫原性的直接影响。方法:自大鼠骨髓分离DC前体细胞于体外诱导、扩增获得成熟DC。C6细胞经PDT处理后,对尚贴壁的细胞以酸洗法获取其表面抗原,从上清液中提取全细胞抗原,未作PDT处理的C6细胞以酸洗或冻融法提取抗原。分别以这4种抗原致敏DC,制成DC疫苗接种SD大鼠后,取脾作CTL的体外杀伤活性测定。结果:用PDT处理后酸洗法获得的抗原刺激DC成熟的能力最强,它制备的DC疫苗诱导的CTL的体外杀伤率最高,为(95.5±1.6)%,而光照后上清组为(90.2±2.4)%,酸洗组为(73.3±2.7)%,冻融组为(63.6±4.9)%,PBS对照组为0。结论:PDT能直接增强肿瘤细胞的免疫原性,用酸洗法获取PDT处理后的肿瘤细胞的抗原肽制备DC疫苗是一种有前景的新方法。

Objective： To investigate the killing activity of cytotoxic T lymphocytes （CTLs） stimulated by dendritic cells （DCs） loaded with C 6 glioma antigen eluted by mild acid after photodynamic therapy（PDT） and test the direct influence of PDT on the immunogenicity of C6 glioma cells. Methods： Progenitors of DCs were isolated from rat bone marrow and cultured with rat recombinant interleukin-4 and rat recombinant granulocyte-macrophage colony stimulating factor to differentiate mature DC. After PDT, the surface antigen peptides were eluted by mild acid from the still adherent C 6 cells and the whole cell antigens were extracted from the supematant. For the PDT-untreated C 6 cells, tumor antigens were made by freeze/thaw or mild acid elution method. Then the four types of antigens were used to pulse DCs to generate DC vaccines. SD rats were inoculated with the four DC vaccines to prepare antigen-specific CTLs. The killing activities of spleen CTLs were determined in vitro. Results： The mild acid-eluted surface antigens from PDT-treated C6 cells promoted the maturation of DCs most efficiently. CTLs stimulated by the DC vaccine had highest cell-killing activity in vitro （95.5 ± 1.6 ） %. The cell-killing rate was （90.2 ± 2.4） % for the CTLs pulsed by whole cell antigens from supematant of PDT-treated C 6 glioma cells, （73.3 ± 2.7 ）% for the CTLs pulsed by acid-eluted antigen from PDF-untreated C 6 glioma cells, （63.6 ± 4.9 ）% for the CTLs pulsed by antigen from PDF-untreated and frozen-thawed C 6 glioma cells, and 0 for PBS control group. Conclusion： PDT directly enhancs immunogenicity of tumor cells. The DC vaccine generated by the antigen peptides eluted by mild acid from PDT-treated tumor cells has a wide prospect of application and research values.