茯苓多糖PCP-Ⅰ对乙肝疫苗抗原的佐剂活性

Adjuvant effect of a polysaccharide PCP-Ⅰ from Poria cocos on HBs Ag antigen in mice

目的研究茯苓多糖（PCP）-Ⅰ对乙肝疫苗抗原免疫小鼠免疫反应的影响。方法采用60℃水提、醇沉、透析和冷冻干燥获得茯苓粗多糖PCP。PCP再经过DEAE-纤维素和Sephadex G-100柱色谱分离,获得相对分子质量均一多糖PCP-Ⅰ。凝胶过滤色谱法测定其峰高相对分子质量,毛细管电泳法测定其单糖组成。乙肝疫苗抗原（HBs Ag,2μg/小鼠）分别与2个剂量PCP-Ⅰ（200μg/小鼠和50μg/小鼠）联用,肌内注射途径免疫小鼠2次,分别于初次免疫后14 d和第2次免疫后14、30、45、60和90 d,采用ELISA法测定小鼠血清特异性抗体Ig G、Ig M、Ig A及Ig G亚类抗体滴度水平。结果 PCP-Ⅰ为相对分子质量均一多糖,峰高相对分子质量约为3.0×104,单糖摩尔比为岩藻糖∶甘露糖∶葡萄糖∶半乳糖=1.00∶1.81∶0.27∶7.27。佐剂活性结果表明,在第2次免疫后14~90 d期间,与单独抗原组相比,联用50或200μg PCP-I均能显著提高受免小鼠血清特异性抗体滴度水平。单独抗原免疫产生的抗体类型主要为Ig G1和Ig M,而联用PCP-I则还产生高滴度的Ig G2a、Ig G2b和Ig G3。PCP-I对乙肝抗原佐剂效果明显优于铝佐剂,而且随着时间的延长,抗体水平变化不明显。PCP-I还能促进免疫小鼠脾T细胞和B细胞增殖,升高CD3＋/CD19＋的比值,降低CD4＋/CD8＋的比值。结论茯苓多糖PCP-I能显著提高乙肝抗原免疫小鼠体液免疫和细胞免疫能力,效果优于铝佐剂。

Objection To investigate the adjuvant activity of a polysaccharide PCP-Ⅰon immune response in HBs Ag immunized mice. Methods Crude polysaccharides from Poria cocos（PCP）were obtained successively by extracting with water at 60℃,ethanol precipitating and dialyzing. A polysaccharide PCP-Ⅰwas separated from PCP by DEAE-cellulose and Sephadex G-100 columns. The sugar content was determined by phenol-sulfuric acid method,monosaccharide compositions were analyzed with capillary electrophoresis,and molecular mass of PCP-Ⅰ was determined with gel filtration chromatography. BALB/c mice were immunized intramuscularly twice with HBs Ag（2 μg/mouse）plus PCP-Ⅰ（50 or 200 μg/mouse）. The serum Ig G and its isotypes antibody titers of immunized mice were analyzed by ELISA at 14,30,45,60 and 90 d after second immunization. The proliferation activities of spleen T and B lymphocytes were determined with MTT method. The populations of CD3＋,CD19＋,CD4＋and CD8＋lymphocytes were determined by flow cytometry. The phagocytosis function of macrophages was evaluated with neutral red method in vitro. Results A new polysaccharide（PCP-Ⅰ）was separated from sclerotium of P. cocos. PCP-I had a relative molecular mass of 3.0×104and was composed of fucose,mannose,glucose and galactose in molar ration of 1.00∶1.81∶0.27∶7.27,respectively. Pharmacological data demonstrated that PCP-Ⅰ significantly increased antigen-specific antibody levels of Ig G1,Ig M,Ig G2 a,Ig G2 b and Ig G3 in immunized mice and generated robust and durable immunity compared to mice immunized with HBs Ag-alum after second immunization for 14~ 90 d. PCP-Ⅰalso improved proliferation of splenocytes,raised the ration of CD3＋/CD19＋cells and decreased the ratios of CD4＋/CD8＋T lymphocytes.PCP-Ⅰ stimulated phagocytosis function of peritoneal macrophages in immunized mice. Conclusion These results suggested that PCP-Ⅰ enhanced humoral and cellular immunity in HBs Ag-immunized mice,and its adjuvant activity was stronger than alum.