不同细胞因子对DC成熟的影响及细胞穿膜肽的穿膜效率比较

目的 通过比较不同因子促进树突状细胞(Dendritic cells,DC)成熟的差别,探讨如何提高DC成熟度。进一步研究多种细胞穿膜肽(Cell penetrating peptide,CPP)的入胞效率,寻找高效的CPP负载肿瘤抗原,以提高DC靶向治疗肿瘤的效果。方法 针对体外培养的DC细胞,应用不同种类、不同浓度的DC培养因子和成熟因子分别诱导DC成熟,通过倒置显微镜观察成熟DC细胞数量、形态;流式检测成熟DC细胞表达CD80、CD83、CD86、HLA-DR的水平。免疫荧光染色观察TAT、SecPen、PanPro和Pan四种细胞穿膜肽穿膜效率的异同。结果 常量及倍量GM-CSF诱导成熟DC的数量无统计学差异(P＞0.05),CD80、CD83、CD86、HLA-DR的表达无统计学差异;应用改良方案培养的成熟DC数量明显高于采用传统IL-4+GM-CSF及IFN-γ诱导的成熟DC数量,差异具有统计学意义(P＜0.05),但是,成熟DC细胞CD80、CD83、CD86、HLA-DR等表面标记表达无统计学差异;SecPen的穿膜效率(83.3±3.0%)明显高于其他三种穿膜肽,具有统计学意义(P＜0.05)。结论 改变GM-CSF的浓度并不能提高成熟DC的数量和比例;相比传统方法,改良方法诱导的DC成熟能力比传统方法有优越性;穿膜肽可以有效进入DC细胞,但不同穿膜肽穿透细胞膜的能力有显著差异,在DC肿瘤疫苗的靶向治疗中需要谨慎选择。

Impacts of the different cytokines on maturation of dendritic cell and the penetrating efficacies of the different cell penetrating peptides

Objective To explore how to improve the mature dendritic cells,we compared the differences between cytokines in promoting the maturation of dendritic cells.We next evaluated the efficacies of several cell penetrating peptides in order to find a high performance CPP with tumor antigens,in order to improve the effectiveness of DC targeted therapy.Methods Several cytokines in different working concentration were in vitro co-cultured with monocytes to induce maturation of dendritic cell.Number and phenotype of the mature dendritic cell were assessed with phase contrast microscope.Surface markers including CD80,CD83,CD86,and HLA-DR were evaluated with flow cytometry.Immunofluorescent staining was used to evaluate the penetrating efficacies of the four peptides TAT,SecPen,PanPro,and Pan.Results GM-CSF in 2 folds concentration didn′t results in increased number of mature dendritic cell when compared with that in conventional concentration(P＞0.05).No statistical significances were found in the expression level of CD80,CD83,CD86,and HLA-DR between the two working concentrations.Compared with conventional method using IL-4,GM-CSF and IFN-γ,number but not functional markers of mature DC was significantly higher when cultured with optimal method(P＜0.05).In comparation with the other three CPPs,SecPen(83.3±3.0%)possess the highest efficacy of internalizing effect(P＜0.05).Conclusion Different concentration of GM-CSF can not increase the number and proportion of mature DC.Compared to the traditional methods,the optimized method is superior in inducing DC maturation than the conventional method.Cell penetrating peptides can efficiently enter DC.However,their penetrating capacity varies significantly.Care must be taken to select the feasible CPP for application in DC vaccines.