Flt3L及CCL5对prime-boost免疫策略中抗原特异性免疫应答增强及.docVIP

Flt3L及CCL5对prime/boost免疫策略中抗原特异性免疫应答增强及 　作者：刘春燕, 郑龙, 尤红煜, 张艳, 王俊霞, 宋淑霞 【摘要】 目的: 研究Flt3L与CCL5作为联合佐剂在prime/boost免疫策略中对HBc抗原特异性免疫应答的增强及抗肿瘤作用。方法: 将两种细胞因子质粒与携带HBc抗原的DNA疫苗经肌内注射法共免疫小鼠, 免疫3次后再用原核表达的HBc颗粒蛋白或HBc DNA疫苗加强, 观察对稳定表达HBcAg 的小鼠黑色素瘤细胞（B16HBc）的生长抑制作用; 并分别采用MTT法检测荷瘤小鼠脾淋巴细胞增殖、 流式细胞术检测脾CD8+T淋巴细胞中IFNγ表达、 ELISA法检测脾淋巴细胞培养上清IL2、 IL4含量及乳酸脱氢酶(LDH)释放法检测特异性CTL杀伤活性。结果: 与对照组相比, 佐剂联合DNA疫苗免疫经蛋白加强组（DDP/Adj）显著抑制肿瘤生长; 佐剂联合DNA疫苗免疫组（DDD/Adj）及DDP/Adj组均可促进特异性淋巴细胞增殖反应（Plt;0.05）, 且DDP/Adj 高于DDD/Adj组（Plt;0.05）; DDD/Adj 及DDP/Adj组小鼠脾脏CD8+T淋巴细胞中IFNγ表达、 IL2 表达水平及CTL杀靶活性均高于对照组(Plt;0.01或Plt;0.05), IL4 表达水平在各组无显著区别（Pgt;0.05）。结论: 在prime/boost免疫策略中, 采用Flt3L与CCL5两种细胞因子联合应用可显著促进荷瘤小鼠产生抗原特异性免疫应答及抗肿瘤作用。 【关键词】 DNA疫苗; Flt3L/CCL5/佐剂; 小鼠; 抗原特异性免疫应答; prime/boost策略 ［Abstract］ AIM: To investigate the immune enhancment and antitumor effect of the recombinant plasmids pFlt3L and pCCL5 in DNA prime/protein boost regimens. METHODS: The mice were coimmunized with HBcAg DNA vaccine and the two cytokines DNA constructs by intramuscular injection for three times at an interval of 2 weeks. Then the mice were boosted with HBc particle proteins or DNA vaccines, respectively. The immune efficacy was evaluated by tumor growth curve. To further investigate the mechanism of inhibiting tumor growth, lymphocytes proliferation response and the number of IFNγproducing cells in splenocytes were measured by MTT or flow cytometry, respectively. The levels of IL2 and IL4 in supernatant of splenolymphocyte cultures were measured by ELISA. The CTL activity of splenolymphocyte was detected with LDH release assay. RESULTS: Compared with negative control, DDP/Adj group significantly inhibit tumor growth; splenocytes proliferation response and the numbers of IFNγproducing cells in DDD/Adj group and DDP/Adj group were significantly higher (Plt;0.05 or Plt;0.01). The levels of IL2 in supernatant of splenolymphocyte cultures in DDD/Adj group and DDP/Adj group were also markedly higher than that of negative control (P