怀牛膝果寡糖对H1N1流感疫苗佐剂活性及免疫细胞功能的影响

摘要

OBJECTIVE To investigate chemical properties of a fructooligosaccharide (ABP-50-FOS)separated from Achyranthes bidentata and immune response in mice immunized H1N1 influenza vaccine. METHODS The methods of GPC,CE,IR and NMR were used to study chemical properties of ABP-50-FOS. BALB/c mice were immunized intramuscularly twice with H1N1 influenza vaccine (3 μg)plus ABP-50-FOS(200 μg)each mouse. The serum total antibody titer and its isotypes titers were analyzed by ELISA. The populations of CD4+,CD8+,CD3+and CD19+lymphocytes were deter⁃mined by flow cytometry. The proliferation activities of spleen T and B lymphocytes were determined with MTT method. The levels of cytokines interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),inter⁃leukin-4(IL-4),IL-12 and NO were measured by ELISA kits. RESULTS ABP-50-FOS was a fructooli⁃gosaccharide with moleculer mass 1885 u. Its bone linkages contained 1,2-and 1,6-fructose residues. ABP-50-FOS could induce high specific-IgG,IgG1,IgG2a,IgG2b and IgM titers after immunization with H1N1 influenza antigen twice(P<0.01). ABP-50-FOS significantly elevated the percentage of CD3+,CD4+ and CD8+ spleen lymphocytes and IFN-γ secretions(P<0.01)in vitro. It also stimulated peritoneal macrophage of mice and DC2.4 dendritic cells to produce TNF-αand IL-12p70 respectively (P<0.01). However,ABP-50-FOS inhibited secretions of NO in macrophage. CONCLUSION The fruc⁃tooligosaccharide ABP-50-FOS separated from A. bidentata can exhibit strong adjuvant activity for H1N1 influenza vaccine.%目的：研究牛膝果寡糖ABP-50-FOS对H1N1流感疫苗的佐剂活性及免疫细胞功能的影响。方法采用凝胶渗透色谱、毛细管电泳、红外光谱和核磁共振等方法研究ABP-50-FOS的理化性质；BALB/c小鼠肌肉注射H1N1流感疫苗3μg和ABP-50-FOS 200μg初次免疫，28 d后加强免疫。分别于初次和再次免疫后14 d用ELISA法检测小鼠血清特异性IgG，IgG1，IgG2a，IgG2b，IgG3和IgM抗体水平；流式细胞仪测定脾细胞中CD3+，CD19+，CD4+和CD8+淋巴细胞亚群百分率；MTT法测定小鼠脾细胞增殖活性；ELISA法测定脾细胞培养上清干扰素γ（IFN-γ）、胸腺细胞培养上清白细胞介素4（IL-4）、腹腔巨噬细胞培养上清肿瘤坏死因子α（TNF-α）和一氧化氮及小鼠骨髓细胞和DC2.4树突状细胞培养上清IL-12p70含量。结果ABP-50-FOS是一种果寡糖，峰高分子质量为1885 u，骨架结构为1，2-连接和1，6-连接的果糖。该寡糖能明显升高H1N1流感疫苗免疫小鼠血清特异性IgG，IgG2a和IgM抗体水平（P<0.01），提高免疫小鼠脾CD3+，CD4+和CD8+T淋巴细胞亚群的百分率。体外实验表明，ABP-50-FOS能促进小鼠脾细胞增殖反应和IFN-γ分泌（P<0.01），促进胸腺细胞IL-4分泌（P<0.01），刺激腹腔巨噬细胞分泌TNF-α（P<0.01）和DC2.4树突状细胞分泌IL-12p70（P<0.01），对巨噬细胞一氧化氮的分泌有抑制作用。结论牛膝果寡糖ABP-50-FOS对H1N1流感疫苗具有良好的佐剂活性，体外对小鼠免疫细胞功能具有促进作用。