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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Ig G,Ig G1,Ig G2a,Ig G2b,Ig G3和Ig M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Ig G,Ig G2a和Ig M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。
Objective To study the effect of ABP-50-FOS on adjuvant activity and immune cell function of H1N1 influenza vaccine. Methods The physicochemical properties of ABP-50-FOS were studied by gel permeation chromatography, capillary electrophoresis, infrared spectroscopy and nuclear magnetic resonance. Primary immunization with 3 μg of H1N1 influenza vaccine and 200 μg of ABP-50-FOS was intranasal in BALB / c mice. After strengthening the immune. Serum specific Ig G, Ig G1, Ig G2a, Ig G2b, Ig G3 and Ig M antibody levels of mice were detected by ELISA method 14 days after primary and secondary immunization respectively. Flow cytometry was used to detect the levels of CD3 +, CD19 + The percentage of CD4 + and CD8 + lymphocyte subsets were determined. The proliferation activity of mouse spleen cells was measured by MTT assay. The levels of interferon-γ (IFN-γ) in supernatant of spleen cells and IL-4 in thymocytes were measured by ELISA. , Peritoneal macrophage culture supernatant tumor necrosis factor alpha (TNF-α) and nitric oxide and mouse bone marrow cells and DC2.4 dendritic cell culture supernatant IL-12p70 content. Results ABP-50-FOS was a fructo-oligosaccharide with a peak molecular mass of 1885 u and a backbone structure of 1,2-linked and 1,6-linked fructose. The oligosaccharides could significantly increase the antibody levels of serum-specific Ig G, Ig G2a and Ig M of mice immunized with H1N1 influenza vaccine (P <0.01) and increase the percentage of splenic CD3 +, CD4 + and CD8 + T lymphocyte subsets . In vitro experiments showed that ABP-50-FOS could promote the spleen cell proliferation reaction and IFN-γ secretion (P <0.01), promote the secretion of IL-4 in thymocytes (P <0.01), and stimulate the secretion of TNF- α by peritoneal macrophages (P <0.01) and DC2.4 dendritic cells secreted IL-12p70 (P <0.01), and inhibited the secretion of nitric oxide in macrophages. Conclusion Achyranthes oligosaccharides ABP-50-FOS has good adjuvant activity on the H1N1 influenza vaccine and can promote the function of immune cells in mice.