摘要
目的探讨共表达人CD40L和IL-2的5/F35嵌合型腺病毒载体(Ad5/F35 CD40L-IL-2),对人单核细胞来源的树突状细胞(Mo-DC)的感染效率及感染后对Mo-DC表型及IL-12产生的影响。方法提取人外周血单个核细胞(PBMCs)总RNA,应用RT-PCR方法克隆CD40L和IL-2,构建Ad5/F35 CD40L-IL-2腺病毒载体。分离和培养人Mo-DC后,应用流式细胞仪分析嵌合型腺病毒载体对Mo-DC的感染效率、CD40L基因表达及感染前后Mo-DC表型的变化;采用ELISA法测定Mo-DC培养上清中细胞因子IL-2和IL-12的含量。结果成功克隆CD40L和IL-2基因并构建成5/F35嵌合型腺病毒载体。Ad5/F35嵌合型腺病毒载体能高效感染Mo-DC,感染效率可达75%以上,Ad5/F35 CD40L-IL-2感染Mo-DC能有效地表达CD40L和分泌IL-2。感染前Mo-DC中CD80、CD86、CD40和HLA-DR呈一定水平表达,而CD83表达水平较低,感染24 h后,CD80、CD86、CD40和HLA-DR表达水平明显上调,特别是CD83。感染不同时间点测定Mo-DC上清IL-12水平,上清中IL-12水平升高呈时间依赖性。结论 Ad5/F35 CD40L-IL-2能高效感染人Mo-DC并表达相应的基因产物,促进Mo-DC成熟和刺激IL-12的分泌。
Objective To investigate Ad5/F35 that coexpressing human CD4OL and interleukin-2（IL-2）,its infection efficiency to human monocyte-derived dendritic cells（Mo-DC）,and its influence on IL-12 production and phenotype changes of Mo-DC.Methods CD40L and IL-2 were cloned by RT-PCR from the total RNA extracted from human peripheral blood mononuclear cells（PBMCs）,and they were used to construct Ad5/F35 CD40L-IL-2.The infection efficiency,expression of CD40L and phenotype changes of Mo-DC infected by Ad5/F35 CD40L-IL-2 were analysed by flow cytometry.Amounts of IL-2 and IL-12 in the supernatants of Mo-DC after the infection of Ad5/F35 CD40L-IL-2 were measured by ELISA.Results The CD40L and IL-2 genes were successfully cloned and the Ad5/F35 CD40L-IL-2 was constructed.Ad5/F35 CD40L-IL-2 efficiently infected Mo-DC by more than 75% infection efficiency,and the infected Mo-DC could efficiently express CD40L and secrete IL-2.The expressions of CD80,CD86,CD40,and HLA-DR on Mo-DC were moderate and CD83 was low before the infection of Ad5/F35 CD40L-IL-2.Those molecules,especially CD83,were markedly upregulated 24 h after the infection.Increasing amounts of IL-12 in the supernatants were detected after the infection at different times in a time-dependent manner.Conclusion Ad5/F35 CD40L-IL-2 can efficiently infect human Mo-DC and express genes.In addition,it stimulates the maturation of Mo-DC and high level of IL-12 production.
出处
《实用癌症杂志》
2013年第3期221-226,共6页
The Practical Journal of Cancer
基金
国家自然科学基金(81172162)
