两种佐剂甘草酸二铵和苦参素对甲型肝炎病毒抗原诱导小鼠体液免疫应答的影响
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摘要
目的探讨两种佐剂甘草酸二铵和苦参素对甲型肝炎病毒(hepatitis A virus,HAV)抗原诱导小鼠体液免疫应答的影响。方法将两种佐剂均按低(100μg)、中(500μg)、高(1 mg)3个剂量,分别与18 EU HAV抗原混合,免疫ICR小鼠,并设生理盐水对照组(生理盐水200μl)、HAV抗原对照组(HAV抗原18 EU)和铝佐剂对照组(HAV抗原18 EU+铝佐剂300μg),共9组,均经腹部皮下多点注射,共免疫1次。分别于免疫后第4、8、12、16周采血,分离血清,采用ELISA法检测抗-HAV IgG抗体水平。免疫后第16周处死小鼠,分别取甘草酸二铵和苦参素最佳剂量组及生理盐水对照组小鼠的心脏、肝脏、脾脏、肺脏、肾脏组织,进行病理观察。结果免疫后第4、8、12、16周,除生理盐水对照组外,其他各组小鼠血清中均可检测到抗-HAV IgG抗体,且抗体水平除甘草酸二铵中剂量组和苦参素低剂量组外,均在第8周达峰值;甘草酸二铵各剂量组小鼠的血清抗体水平均明显高于HAV抗原对照组(P<0.05),其中甘草酸二铵低剂量组的抗体水平最高,且在第8周时超过铝佐剂对照组;苦参素各剂量组小鼠免疫后各周血清抗-HAV IgG抗体水平均显著高于HAV抗原对照组(P<0.05),其中苦参素低剂量组抗体水平最高,其抗体水平在第12周达峰值。实验期间,各组小鼠均未出现异常表现,各组织均未发现病理改变。结论甘草酸二铵和苦参素均能明显增强HAV抗原诱导小鼠体液免疫应答的水平,具有成为新型疫苗佐剂的研发价值。
Objective To investigate the effect of diammonium glycyrrhizinate and oxymatrine adjuvant on humoral immune response induced by hepatitis A virus(HAV)antigen in mice. Methods The two kinds of adjuvant,each at low(100 μg),moderate(500 μg)and high(1 mg)dosages,were mixed with 18 EU HAV antigen separately and immunized s. c. to ICR mice in several sites,using 200 μl physiological saline,HAV antigen(18 EU)and aluminum adjuvant(300 μg aluminum adjuvant + 18 EU HAV antigen)as controls,nine groups in total. Serum samples were collected at weeks 4,8,12 and 16 after immunization and determined for anti-HAV IgG levels by ELISA. The mice were killed at week 16 after immunization,and the heart,liver,spleen,lung and kidney tissues of mice immunized with diammonium glycyrrhizinate and oxymatrine adjuvant at optimal dosages as well as those in physiological saline control group were subjected to pathological examination. Results Anti-HAV IgGs were detected in the sera of mice in various groups except physiological saline control group. The IgG levels,excepted those in diammonium glycyrrhizinate moderate dosage and oxymatrine low dosage groups,reached the maximums at weeks 8. The serum anti-HAV IgG levels of mice immunized with diammonium glycyrrhizinate at various dosages were significantly higher than that in HAV antigen control group(P < 0. 05),of which that in diammonium glycyrrhizinate low dosage group was the highest and was higher than that in aluminum adjuvant control group at week 8. However,the serum IgG levels of mice in oxymatrine groups at various weeks were significantly higher than those in HAV antigen group(P < 0. 05),of which that in oxymatrine low dosage group was the highest and reached the maximum at week 12. No abnormalities were observed during the whole study,and no pathological changes were observed in various tissues. Conclusion Both diammonium glycyrrhizinate and oxymatrine adjuvant enhanced the humoral immune responses induced by HAV antigen in mice,which might be developed as novel adjuvant of vaccines.
Objective To investigate the effect of diammonium glycyrrhizinate and oxymatrine adjuvant on humoral immune response induced by hepatitis A virus(HAV)antigen in mice. Methods The two kinds of adjuvant,each at low(100 μg),moderate(500 μg)and high(1 mg)dosages,were mixed with 18 EU HAV antigen separately and immunized s. c. to ICR mice in several sites,using 200 μl physiological saline,HAV antigen(18 EU)and aluminum adjuvant(300 μg aluminum adjuvant + 18 EU HAV antigen)as controls,nine groups in total. Serum samples were collected at weeks 4,8,12 and 16 after immunization and determined for anti-HAV IgG levels by ELISA. The mice were killed at week 16 after immunization,and the heart,liver,spleen,lung and kidney tissues of mice immunized with diammonium glycyrrhizinate and oxymatrine adjuvant at optimal dosages as well as those in physiological saline control group were subjected to pathological examination. Results Anti-HAV IgGs were detected in the sera of mice in various groups except physiological saline control group. The IgG levels,excepted those in diammonium glycyrrhizinate moderate dosage and oxymatrine low dosage groups,reached the maximums at weeks 8. The serum anti-HAV IgG levels of mice immunized with diammonium glycyrrhizinate at various dosages were significantly higher than that in HAV antigen control group(P < 0. 05),of which that in diammonium glycyrrhizinate low dosage group was the highest and was higher than that in aluminum adjuvant control group at week 8. However,the serum IgG levels of mice in oxymatrine groups at various weeks were significantly higher than those in HAV antigen group(P < 0. 05),of which that in oxymatrine low dosage group was the highest and reached the maximum at week 12. No abnormalities were observed during the whole study,and no pathological changes were observed in various tissues. Conclusion Both diammonium glycyrrhizinate and oxymatrine adjuvant enhanced the humoral immune responses induced by HAV antigen in mice,which might be developed as novel adjuvant of vaccines.
引文
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[11]Chen Y,Zheng CW,Chen GJ,et al.Adjuvant effect of ginsenoside Rg1[J].Bull Acad Mil Med Sci,2009,33(3):251-253,292.(in Chinese)陈宇,郑纯威,陈国江,等.人参皂苷Rg1免疫佐剂作用的研究[J].军事医学科学院院刊,2009,33(3):251-253,292.
[12]Xue R,Zhang J,Cao JH,et al.Effect on immunological function of isatis indigotica polysaccharide in mice[J].Guid J Trad Chin Med Pharm,2012,18(9):94-96.(in Chinese)薛瑞,章激,曹军华,等.板蓝根多糖对小鼠免疫功能的调节作用[J].中医药导报,2012,18(9):94-96.
[2]Vecchi S,Bufali S,Skibinski DA,et al.Aluminum adjuvant dose guidelines in vaccine formulation for preclinical evaluations[J].J Pharm Sci,2012,101(1):17-20.
[3]Manns MP,Wedemeyer H,Singer A,et al.Glycyrrhizin in patients who failed previous interferon alpha-based therapies:biochemical and histological effects after 52 weeks[J].J Viral Hepat,2012,19(8):537-546.
[4]Fang HY,Song XL,Wu XM.Comparison of effects of oral and parenteral diammonium glycyrrhizinate on some immune function in mice[J].Chin J MAP,2002,19(4):274-275.(in Chinese)方红英,宋小莉,吴锡铭.口服和注射甘草酸二铵对小鼠的免疫功能影响的比较[J].中国现代应用药学杂志,2002,19(4):274-275.
[5]Zhang JF,Li H,Li CQ,et al.Effect of diammonium glycyrrhizinate on proliferation in liver cancer cell SMMC-7721 and p53 expression[J].Chongqing J Med,2012,41(27):2852-2853,2856.(in Chinese)张剑锋,李浩,李超乾,等.甘草酸二铵对人肝癌细胞SMMC-7721增殖和p53表达的影响[J].重庆医学,2012,41(27):2852-2853,2856.
[6]Ye JY,Yin ZB,Guan MQ,et al.Role of glycyrrhizin in the treatment of pulmonary fibrosis in rats and impact of this agent on serum IL4/IFN-γlevel[J].J Pract Med,2011,27(6):960-962.(in Chinese)叶进燕,殷宗宝,管敏强,等.甘利欣对大鼠肺纤维化的治疗作用及对血清IL4/IFN-γ浓度的影响[J].实用医学杂志,2011,27(6):960-962.
[7]Lu X,Lin B,Tang JG,et al.Study on the inhibitory effect of total alkaloids of sophora alopecuroides on osteosarcoma cell growth[J].Afr J Tradit Complement Altern Med,2013,11(1):172-175.
[8]Zhang D,Liu S,Yao M,et al.Effects of vietnamese sophora root on growth,adhesion,invasion and motility of melanoma cells[J].Afr J Tradit Complement Altern Med,2013,11(1):62-66.
[9]Shaw CA,Tomljenovic L.Aluminum in the central nervous system(CNS):toxicity in humans and animals,vaccine adjuvants,and autoimmunity[J].Immunol Res,2013,56(2-3):304-316.
[10]Vera-Lastra O,Medina G,Cruz-Dominguez Mdel P,et al.Autoimmune/inflammatory syndrome induced by adjuvants(Shoenfeld′s syndrome):clinical and immunological spectrum[J].Expert Rev Clin Immunol,2013,9(4):361-373.
[11]Chen Y,Zheng CW,Chen GJ,et al.Adjuvant effect of ginsenoside Rg1[J].Bull Acad Mil Med Sci,2009,33(3):251-253,292.(in Chinese)陈宇,郑纯威,陈国江,等.人参皂苷Rg1免疫佐剂作用的研究[J].军事医学科学院院刊,2009,33(3):251-253,292.
[12]Xue R,Zhang J,Cao JH,et al.Effect on immunological function of isatis indigotica polysaccharide in mice[J].Guid J Trad Chin Med Pharm,2012,18(9):94-96.(in Chinese)薛瑞,章激,曹军华,等.板蓝根多糖对小鼠免疫功能的调节作用[J].中医药导报,2012,18(9):94-96.