板蓝根α-葡聚糖佐剂提高H1N1流感疫苗免疫小鼠体液免疫和细胞免疫功能  

Effect of α-glycan isolated from Isatis indigotica on humoral and cellular immunities in mice immunized with H1N1 influenza vaccine

作　　者：文松[1] 李倩[2] 贾培媛[2] 赵修南[2] 武军华[2] 麻浩[2] 张永平 王玉霞[2] 单俊杰[2] 王海南[1,4] 

机构地区：[1]贵州大学药学院,贵州贵阳550025 [2]军事医学科学院毒物药物研究所,北京100850 [3]北京中安佐际生物科技有限公司,北京100111 [4]国家食品药品监督管理总局,北京100053

出　　处：《中国药理学与毒理学杂志》2014年第6期850-856,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：国家新药创制重大专项(2012ZX09301003-001);国家新药创制重大专项(2013ZX09402103);国家科技攻关计划(2011BAD26B02-3)~~

摘　　要：目的：研究板蓝根α-葡聚糖对H1N1流感疫苗免疫小鼠体液免疫和细胞免疫功能的影响。方法100μg α-葡聚糖按＋H1N1流感病毒裂解液3μg给每只小鼠一次性肌内注射。分别在免疫后3,5,8,10,12和14 d采血,ELlSA法检测小鼠血清特异性抗体和亚类抗体滴度；MTT法测定小鼠脾T淋巴细胞、B淋巴细胞和巨噬细胞增殖反应；ELlSA法测定细胞培养上清干扰素γ（ lFN-γ）、白细胞介素4（ lL-4）、lL-12和肿瘤坏死因子-α（ TNF-α）含量；流式细胞术测定脾细胞中CD3^＋, CD19^＋, CD4^＋, CD8^＋T细胞亚群百分率。结果板蓝根α-葡聚糖对人用H1N1流感疫苗抗原显示优良的佐剂作用,初次免疫后5 d即能促进特异性抗体的生成,抗体亚型主要为lgM。免疫后5～14 d,总抗体及亚类lgG1, lgG2a和lgG2b滴度持续升高,lgG3在免疫8 d达峰,之后无明显变化。 lgA滴度水平最低,且随时间无明显变化。α-葡聚糖显著促进H1N1流感疫苗免疫小鼠脾T 细胞和B 细胞增殖（增殖率分别为44.2％和37.8％）,促进脾细胞分泌 lFN-γ（ P＜0.01）和lL-12（ P＜0.05）,刺激胸腺细胞分泌lL-4（ P＜0.01）；与单独抗原组相比,联用α-葡聚糖明显提高免疫小鼠脾 CD3^＋细胞百分率（P＜0.01）及 CD3＋/CD19＋比值（P＜0.01）,提高 CD8^＋T 细胞百分率,降低CD4^＋/CD8^＋比值（ P＜0.01）。α-葡聚糖能激活巨噬细胞,促进TNF-α的分泌。结论板蓝根α-葡聚糖能显著提高免疫小鼠的体液免疫和细胞免疫功能。OBJECTlVE To investigate the effect of α-glycan isolated from Isatis indigotica on humoral immunity and cellular immunity functions in mice immunized with H1N1 influenza vaccine. METHODS BALB/c mice were immunized intramuscularly once with H1N1 influenza vaccine （ 3 μg） plusα-glycan （ 100μg） each mouse. The serum total antibody titer and its isotype antibody titer of immu-nized mice were analyzed by ELlSA at 5, 8, 10, 12 and 14 d after injection at vaccine. The proliferation activities of spleen T and B lymphocytes were determined with MTT method. The levels of cytokines interferon-γ（ lFN-γ） , tumor necrosis factorα（ TNF-α） , interleukin-4（ lL-4） and lL-12 were measured by ELlSA kits. The populations of CD4^＋, CD8^＋, CD3^＋ and CD19^＋ lymphocytes were determined by flow cytometry. Furthermore, the proliferation rate of macrophages was studied with MTT method in vitro. RESULTS The α-glycan from I.indigotica could gradually induce high specific-antibody production 5-14 d after immunization with H1N1 influenza antigen plus theα-glycan in mice compared to immunization with antigen alone （ P〈0.01） . After injection of antigen withα-glycan for 5 d, the main lgG isotype was lgM, and the titer levels of total lgG, lgG1 , lgG2a and lgG2b were also significantly raised following 5-14 d after immunization. The α-glycan significantly promoted the spleen T and B lymphocytes proliferation （ growth rate 44.2%and 37.8%） , stimulated the secretion of lFN-γand lL-12 of splenocytes （ P〈0.01, P〈0.05） , and also promoted lL-4 secretion of thymocytes （P〈0.01）. The polysaccharide significantly raised the percent age of CD3^＋T cells （ P〈0.01） , CD3^＋/CD19^＋ T lymphocytes （ P〈0.01） , and CD8^＋ T cells （ P〈0.01） but decreased the percentage of CD4^＋/CD8^＋ T lymphocytes compared with antigen alone group （ P〈0.01） . Furthermore, the α-glycan exhibited significant effects on the proliferation and TNF-α secre-tion of MH-S macrophages. CONCLUS

关 键 词：板蓝根 α-葡聚糖 流感疫苗 佐剂 免疫 抗体生成 免疫 细胞 

分 类 号：R392[医药卫生—免疫学] R285[医药卫生—基础医学]