摘要
目的探讨DNA四面体骨架(DNA-TH)作为黏膜疫苗载体以及佐剂的可行性。方法利用软件设计DNA-TH,将佐剂CpG序列设计整合于骨架中,体外合成后与SA结合形成DNA四面体纳米颗粒(SA-DNA-TH)。体外实验中,以游离SA和二维结构SA-CpG为对照,检测SA-DNA-TH被小鼠原代巨噬细胞的摄取效率及对抗原呈递细胞(APC)的激活能力。动物实验中,通过口内黏膜下注射,以游离SA与游离CpG混合物(混合组)为对照,采用免疫荧光染色观察小鼠淋巴结内SA分布,并检测血清及唾液中抗SA特异性抗体(血清IgG、IgM;唾液sIgA)水平,评估体液免疫和黏膜免疫效果。结果非变性聚丙烯酰胺凝胶电泳证实DNA-TH及SA-DNA-TH成功合成。体外实验结果显示,SA-DNA-TH可被原代巨噬细胞迅速摄取,其摄取率(92.65%±4.43%)显著高于SA-CpG组(25.37%±3.56%)及游离SA组(1.80%±1.02%),差异均有统计学意义(均P<0.001);SA-DNA-TH激活APC的程度(OD值倍数为3.60±0.32)也显著高于游离SA组(1.13±0.10)及SA-CpG组(1.21±0.02),差异均有统计学意义(均P<0.001)。动物实验结果显示,SA-DNA-TH组SA信号与淋巴结被膜下窦巨噬细胞及树突状细胞信号重叠,而混合组SA呈游离分散信号,不与APC信号重叠;与空白组、混合组相比,SA-DNA-TH组诱导的血清抗SA抗体(IgG+IgM)滴度及唾液抗SA-sIgA抗体滴度均较高(均P<0.05)。结论DNA-TH能有效递送模式抗原SA至呈递细胞,显著诱导血清特异性抗体产生并激活黏膜免疫反应,具有成为黏膜疫苗开发的载体及佐剂的潜力。
ObjectiveTo investigate the feasibility of DNA tetrahedral framework(DNA-TH)as a carrier and adjuvant for mucosal vaccines,using streptavidin(SA)as a model antigen.MethodsDNA-TH was designed using software,integrating the adjuvant CpG sequence into its structure.After in vitro synthesis,it was conjugated with SA to form SA-DNA-TH nanoparticles.In vitro experiments:free SA and the two-dimensional structure SA-CpG(SA directly conjugated to CpG)were used as controls.The uptake efficiency of SA-DNA-TH by mouse primary macrophages and its ability to activate antigen-presenting cells(APCs)were evaluated.In vivo experiments:following submucosal oral injection,a mixture of free SA and free CpG(mixed group)was used as a control.The distribution of SA within mouse lymph nodes was observed using immunofluorescence staining.Levels of SA-specific antibodies(serum IgG,IgM;salivary sIgA)in serum and saliva were measured to assess humoral and mucosal immune responses.ResultsNative polyacrylamide gel electrophoresis confirmed the successful synthesis of DNA-TH and SA-DNA-TH.In vitro experiments:SA-DNA-TH was rapidly taken up by primary macrophages.Its uptake rate(92.65%±4.43%)was significantly higher than that of the SA-CpG group(25.37%±3.56%)and the free SA group(1.80%±1.02%;both P<0.01).SA-DNA-TH also induced significantly stronger APC activation(OD value fold increase:3.60±0.32)compared to the free SA group(1.13±0.10)and the SA-CpG group(1.21±0.02;both P<0.01).In vivo experiments:lymph node distribution analysis revealed overlapping signals of SA with subcapsular sinus macrophages(SCSMs)and dendritic cells(DCs)in the SA-DNA-TH group,whereas SA signals appeared dispersed and non-overlapping with APCs in the mixed group.Regarding immunogenicity,both serum anti-SA antibody(IgG+IgM)titers and salivary anti-SA sIgA antibody titers induced by SA-DNA-TH were significantly higher than those in the mixed group and the blank control group(both P<0.05).ConclusionDNA-TH effectively delivers the model antigen SA to antigen-presenting cells,significantly induces the production of serum-specific antibodies,and activates mucosal immune responses.It demonstrates potential as a carrier and adjuvant for developing mucosal vaccines.
作者
陈萧瞳
杨静
刘恒朗
王莉丽
Chen Xiaotong;Yang Jing;Liu Henglang;Wang Lili(School of Medicine,University of Electronic Science and Technology of China,Chengdu 610031,China;Department of Stomatology,Sichuan Provincial People′s Hospital,University of Electronic Science and Technology of China,Chengdu 610072,China)
出处
《中华预防医学杂志》
北大核心
2025年第8期1270-1278,共9页
Chinese Journal of Preventive Medicine
基金
四川省科技计划(2024NSFSC1881)
四川省医学科学院·四川省人民医院青年人才基金(2021QN06)
四川省医学青年创新科研课题计划(Q23011)。
