橙皮素对mRNA疫苗的免疫增强作用及其机制研究

Immunoenhancement Effect of Hesperetin on mRNA Vaccines and Its Mechanism

目的:探究橙皮素作为mRNA疫苗的佐剂对其肿瘤预防作用产生的影响及其相关免疫机制,为开发中药及其单体成分作为疫苗佐剂提供研究思路。方法:利用微流控技术制备脂质纳米颗粒(lipid nanoparticles,LNPs),使其单独或同时负载mRNA和橙皮素;测定mRNA-LNPs负载橙皮素前后的粒径、包封率;利用流式细胞术等方法考察DC细胞对复合物的摄取与表达情况;将C57BL/6小鼠随机分为PBS对照组(NC组)、橙皮素单药组(Hes组)、编码鸡卵清蛋白(ovalbumin,OVA)的mRNA单药组(mRNA组)和橙皮素-mRNA组(Hes-mRNA组),分别于第0天和第7天皮下注射相应药物进行免疫,于第1次接种28天后,给小鼠皮下接种E.G7 OVA细胞造瘤并监测肿瘤生长趋势和小鼠生存情况;流式细胞术检测小鼠淋巴结中CD4^(+)、CD8^(+)T细胞表达IFN-γ的水平,ELISA检测小鼠血清中OVA特异性抗原IgG(OVA-sIgG)的水平。结果:橙皮素-脂质纳米颗粒系统稳定,能实现mRNA的胞内递送。与NC组相比,Hes组小鼠的生存期延长,差异具有统计学意义(P<0.05),mRNA组和Hes-mRNA组小鼠生存期延长,差异具有统计学意义(P<0.05,P<0.001);与NC组相比,mRNA组和Hes-mRNA组小鼠的肿瘤生长缓慢,差异具有统计学意义(均P<0.05);与mRNA组相比,Hes-mRNA组小鼠肿瘤生长速度和生存期均有显著改善,差异具有统计学意义(均P<0.05)。与NC组相比,mRNA组和Hes-mRNA组小鼠的IFN-γ^(+)CD4^(+)T细胞水平和IFN-γ^(+)CD8^(+)T细胞水平均显著升高(P<0.05)。与NC组相比,Hes组小鼠的血清中OVA-sIgG的含量显著增高(P<0.001)。结论:橙皮素作为中药佐剂可以通过静电吸附作用稳定地加入mRNA-LNPs系统,并且其自身具有肿瘤预防和诱导小鼠体液免疫作用,能显著延长荷瘤小鼠的生存时间,与mRNA联合使用能同时激活机体细胞免疫和体液免疫,有效延缓肿瘤生长,增强肿瘤预防作用。

Objective:To investigate the influence of hesperetin as an adjuvant of mRNA vaccines on tumor prevention effect and its immune mechanism,and provide new ideas for the development of traditional Chinese medicine and its monomer components as vaccine adjuvants.Methods:Lipid nanoparticles(LNPs)loaded with mRNA and/or hesperetin were prepared via microfuidics.Particle size and encapsulation efficiency of complexes were determined once prepared.The uptake and expression of the complexes by dendritic cells were investigated by flow cytometry.C57BL/6 mice were randomly assigned to the phosphate buffer solution control group(NC group),the hesperetin-LNPs group(Hes group),the ovalbumin(OVA)mRNA-LNPs group(mRNA group)and the hesperetin OVA mRNA-LNPs group(Hes-mRNA group),then were immunized by hypodermically injecting with drugs on day 0 and day 7,respectively.E.G7 OVA cells were subcutaneously inoculated 28 days after the first injection.Tumor volume and survival of the mice were monitored.The levels of IFN-γin CD4^(+)and CD8^(+)T cells in the lymph nodes of mice were detected by flow cytometry.The concentration of OVA-sIgG in the serum of mice was detected by ELISA.Results:The delivery system was stable and enables the intracellular delivery of mRNA.The survival time in the Hes group,the mRNA group and the Hes-mRNA group were longer than that in the NC group(P<0.05,P<0.05 and P<0.001);mRNA group and Hes-mRNA group showed a significant delay of tumor growth compared to the NC group(P<0.05).Tumor growth rate and survival in the Hes-mRNA group were significantly higher than those in the mRNA group(P<0.05).Compared with the NC group,the levels of IFN-γ^(+)CD4^(+)and IFN-γ^(+)CD8^(+)T cells significantly increased in the mRNA group and the Hes-mRNA group(P<0.05).Compared with the NC group,the concentration of OVA-sIgG in the serum of mice in the Hes group significantly increased(P<0.001).Conclusion:Hesperetin,as Chinese medicine adjuvant,can be stably added into the mRNA-LNPs system by electrostatic adsorption,and can prevent tumor and induce humoral immunity in mice.The combined use of hesperetin and mRNA can simultaneously activate both cellular immunity and humoral immunity,effectively delay tumor growth,and enhance the effect of tumor prevention.