甲胎蛋白与热休克蛋白70融合DNA疫苗抗肿瘤免疫

Immunotherapy with a chimeric AFP and HSP70 gene DNA vaccine targeting on a marine hepatocellular carcinoma

目的通过小鼠甲胎蛋白(AFP)与结核分枝杆菌热休克蛋白70(HSP70)基因融合制备DNA疫苗,研究该种DNA疫苗诱导小鼠的免疫应答及其对荷瘤小鼠的治疗作用。方法应用分子克隆技术构建AFP与HSP70的融合表达载体,免疫实验共分5组:磷酸盐缓冲液组、pcDNA3．1组、pcDNA3.1-AFP组、pcDNA3．1-HSP70组、pcDNA3．1- AFP-HSP70组(融合疫苗组),每组7只小鼠。2次免疫后分离小鼠脾细胞,体外诱导细胞毒性T淋巴细胞做杀伤实验, 以乳酸脱氢酶法检测杀伤率,以酶联免疫吸附法检测脾细胞释放的干扰素γ;体外培养小鼠肝癌细胞系Hepa1-6,以每200 μl 4×106细胞数在C57BL／6J小鼠右前腋皮下注射作荷瘤鼠模型,以融合疫苗免疫荷瘤鼠,观测肿瘤大小评价融合疫苗的治疗作用。结果AFP与HSP70的融合疫苗能诱导AFP特异性细胞毒性T淋巴细胞反应,HSP70对于该反应有增强作用(P<0．05),杀伤率在效:靶比为50:1时为32％左右;免疫后小鼠脾细胞体外刺激后分泌干扰素γ约200 pg／ml,融合疫苗组分泌高于其他组(P<0．05),融合疫苗组肿瘤小于其他组(P<0．05),生存时间长于其他组(P<0．05)。结论 AFP与HSP70的融合疫苗能激发AFP特异性细胞毒性T淋巴细胞．并且对肝癌移植瘤有治疗作用。

Objective To investigate immune responses and the anti-tumor effect of a constructed Chimeric AFP-Mt. HSP70 DNA vaccine in mice. Methods Chimeric AFP-Mt.HSP70 was constructed by molecular clone techniques. Spleen cells derived from mice immunized twice were induced to secrete IFN γ and were assayed using ELISA, The activity of the cytotoxic lymphocytes （CTL） derived from spleen cells was assayed using lactate dehydrogenase （LDH）. 4 × 10^6 Hepa1-6 cells/200μl were injected subcutaneously into the right axilla of each mouse bearing the tumor. The anti-tumor effect of the recombinant DNA vaccine was evaluated by measuring tumor sizes of the mice, Results AFP-specific CTL reaction was induced by our chimeric DNA,accine and Mt.HSP70 enhanced this effect （P 〈 0.05）. The CTL activity was about 32% at E/T = 50：1. The IFN γ secreted by spleen cells of mice immunized with chimeric plasmids was about 200 pg/ml. It was higher than those in the other groups （P 〈 0.05）; Tumor sizes of mice immunized with fused plasmids were smaller than those in the other groups （P 〈 0.05）. Survival times of mice immunized with the fused plasmids were prolonged. Condusion Chimeric DNA vaccine can induce AFP-specific CTL reaction and has an anti-tumor effect on transplanted tumors in our murine experiment.