MIP-1α和Flt3l基因佐剂联合应用对HPV16E7 DNA疫苗的免疫增强作用

Coadministration of gene adjuvant MIP-1α and Flt3l to enhance the immunological effect of DNA vaccine to HPV16E7

目的:探讨巨噬细胞炎性蛋白1α(Macrophage inflammatory protein-1α,MIP-1α)和酪氨酸激酶受体3配体(Flt3ligand,Flt3l)基因佐剂联合应用增强人乳头瘤病毒16型(Human papillomavirus16,HPV16)E7DNA疫苗免疫效果的可能性。方法:构建真核表达质粒pcDNA3/MIP-1α、pcDNA3/Flt3l和pcDNA3/HPV16E7。C57BL/6小鼠随机分为8组,分别为pcDNA3组、pcDNA3/MIP-1α组、pcDNA3/Flt3l组、pcDNA3/MIP-1α和pcDNA3/Flt3l混合组、pcDNA3/HPV16E7组、pcDNA3/MIP-1α和pcDNA3/HPV16E7混合组、pcDNA3/Flt3l和pcDNA3/HPV16E7混合组以及pcDNA3/MIP-1α、pcDNA3/Flt3l和pcDNA3/HPV16E7混合组。每组14只,肌肉注射质粒免疫小鼠,每3周免疫1次,共3次;末次免疫后2周每组随机抽取6只小鼠,取脾脏制备淋巴细胞悬液,用CCK-8试剂盒检测其对TC-1靶细胞的特异性杀伤能力。其余小鼠在腹股沟处皮下注射5×104个TC-1细胞,观察肿瘤生长情况。结果:成功构建了真核表达质粒pcDNA3/MIP-1α、pcDNA3/Flt3l和pcDNA3/HPV16E7;pcDNA3/MIP-1α、pcDNA3/Flt3l和pcDNA3/HPV16E7混合组的CTL杀伤能力明显高于其它各组(P<0.01);肿瘤细胞攻击后16天,pcDNA3组小鼠全部长出肿瘤,而pcDNA3/MIP-1α、pcDNA3/Flt3l和pcDNA3/HPV16E7混合组的成瘤率仅为12.5%,明显低于其他组,统计学分析示8组之间成瘤率差异有显著性(P=0.007)。60天后各组成瘤率之间的差异无统计学意义(P=0.229)。结论:联合应用MIP-1α和Flt3l基因佐剂增强了HPV16E7DNA疫苗的免疫效果,一定程度上延缓了肿瘤的发生。

Objective：To study the possibility for enhancing the immunological effects of DNA vaccine to HPV16E7 by combination of gene adjuvant macrophage inflammatory protein-1α（MIP-1α） and Flt3 ligand（Flt3l）.Methods：The eukaryotic expressing plasmids,which in-cluded pcDNA3/MIP-1α,pcDNA3/HPV16E7 and pcDNA3/Flt3l.C57BL/6 mice were divided into 8 groups randomly.The groups were pcDNA3 group,pcDNA3/MIP-1α group,pcDNA3/Flt3l group,pcDNA3/MIP-1α and pcDNA3/Flt3l mixed group,pcDNA3/HPV16E7 group,pcDNA3/MIP-1α and pcDNA3/HPV16E7 mixed group,pcDNA3/Flt3l and pcDNA3/HPV16E7 mixed group,pcDNA3/MIP-1α and pcDNA3/Flt3l and pcDNA3/HPV16E7 mixed group,respectively.Each group contained 14 mice.Plasmids were performed to mice by intramuscular injection at 3 week intervals for 3 times.6 mice of each group were sacrificed randomly 2 weeks after the last immunization event with collection of lymphocyte suspension from spleens.Specific cytotoxic T lymphocytes（CTLs） targeting to TC-1 cells were tested by CCK-8 kit.The rest mice were challenged with 5×10^4 TC-1 cells via subcutaneous injection in fold inguen.The growth of tumours was observed.Results：The eukaryotic expressing plasmids of pcDNA3/MIP-1α,pcDNA3/HPV16E7 and pcDNA3/Flt3l were constructed successfully.The CTL level of pcDNA3/MIP-1α and pcDNA3/Flt3l and pcDNA3/HPV16E7 three plasmids mixed groups was higher than those of the other groups remarkably（P〈0.01）.16 days after challenge by tumour cells,all mice of pcDNA3 group formed tumors,while the tumor-forming rate of pcDNA3/MIP-1α and pcDNA3/Flt3l and pcDNA3/HPV16E7 three plasmids mixed group was 12.5%,which was lower than the other groups markedly.The difference of tumor-forming rate among 8 groups was significant by statistics analysis（P=0.007）.60 days after the challenge,the difference among all groups had no statistic significance（P=0.229）.Conclusion：The immunological effect of DNA vaccine to HPV16E7 is enhanced by coadmin-istration of gene adjuvant MIP-1α and Flt3l.They delay the tumor growth to a certain extent.