板蓝根α-葡聚糖佐剂提高H1 N1流感疫苗免疫小鼠体液免疫和细胞免疫功能

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. CONCLUSlON Theα-glycan isolated from I.indigotica can improve humoral and cellular immunity response in mice immunized with H1N1 influenza vaccine.