羟丙基-β-环糊精递送皂苷免疫佐剂效果的初步研究

Preliminary study on the effects of hydroxypropyl-β-cyclodextrin delivery of saponin immunoadjuvant

为了了解羟丙基-β-环糊精(hydroxypropyl-β-cyclodextrin,HP-β-CD)作为递送剂对皂苷类免疫佐剂Quil A的递送效果和佐剂活性的影响,试验将25只6~8周龄健康的雌性Balb/c小鼠随机分为5组,分别为HP-β-CD+Q组、免疫刺激复合物(immunostimulatory complexes,ISCOMs)组、Quil A组、卵清蛋白(OVA)组及PBS组,其中HP-β-CD+Q组、ISCOMs组、Quil A组、OVA组均通过后肢肌肉注射免疫OVA抗原,HP-β-CD+Q组、ISCOMs组、Quil A组分别免疫HP-β-CD+Quil A、ISCOMs、Quil A,免疫时间为第0,21,42天,并于第14,35,56天分别进行采血,分离血清,采用酶联免疫吸附法(ELISA)检测IgG、IgG1和IgG2a抗体水平,MTT法检测脾淋巴细胞的刺激指数,流式细胞术检测免疫后脾脏CD4^(+)、CD19^(+)淋巴细胞亚群的数量变化。结果表明:第三次免疫后,HP-β-CD+Q组血清中OVA特异性抗体IgG质量浓度极显著高于OVA组(P<0.01),Quil A组显著高于OVA组(P<0.05);HP-β-CD+Q组、ISCOMs组血清中OVA特异性抗体IgG1、IgG2a质量浓度极显著高于OVA组(P<0.001或P<0.01),且HP-β-CD+Q组血清中OVA特异性抗体IgG1质量浓度约是ISCOMs组及Quil A组的2倍,约是OVA组的10倍;HP-β-CD+Q组血清中OVA特异性IgG2a质量浓度约是ISCOMs组及Quil A组的1.3倍,约是OVA组的6.5倍。HP-β-CD+Q组和ISCOMs组脾脏淋巴细胞刺激指数显著高于OVA组(P<0.05)。HP-β-CD+Q组和ISCOMs组脾脏CD4^(+)淋巴细胞亚群数量极显著低于OVA组(P<0.01),Quil A组显著低于OVA组(P<0.05);ISCOMs组脾脏CD19^(+)淋巴细胞亚群数量显著低于OVA组(P<0.05)。说明HP-β-CD能够增强Quil A的佐剂活性,刺激机体产生高水平抗体,且有可能通过促进CD4^(+)向外周淋巴组织的迁移介导抗体反应加强。

In order to understand the effects of hydroxypropyl-β-cyclodextrin(HP-β-CD)as a delivery agent on the delivery and adjuvant activity of the saponin-based adjuvant Quil A,25 healthy female Balb/c mice from 6 to 8 weeks of age were randomly divided into 5 groups,namely,HP-β-CD+Q group,immunostimulatory complexes(ISCOMs)group,Quil A group,ovalbumin(OVA)group and PBS group,in which HP-β-CD+Q group,ISCOMs group,Quil A group and OVA group were immunized with OVA antigen by intramuscular injection of hind limbs,and HP-β-CD+Q group,ISCOMs group and Quil A group were immunized with HP-β-CD+Quil A,ISCOMs,Quil A respectively on day 0,21 and 42,and blood was collected and serum was isolated on day 14,35 and 56,respectively.The levels of IgG,IgG1 and IgG2a antibodies produced by the organism after immunization were detected by enzyme-linked immunosorbent assay(ELISA);the stimulation indexes of splenic lymphocytes after immunization were detected by MTT;and the quantitative changes of splenic CD4^(+)and CD19^(+)lymphocyte subpopulations after immunization were detected by flow cytometry.The results showed that the serum mass concentration of OVA-specific antibody IgG in the HP-β-CD+Q group was extremely significantly higher than that in the OVA group(P<0.01),and that in the Quil A group was significantly higher than that in the OVA group(P<0.05);the serum mass concentrations of OVA-specific antibody IgG1 and IgG2a in the HP-β-CD+Q and ISCOMs groups were extremely significantly higher than those in the OVA group(P<0.001 or P<0.01),and the serum mass concentrations of OVA-specific antibodies IgG1 in the HP-β-CD+Q group were about twice as high as those in the ISCOMs and Quil A groups and about 10 times as high as those in the OVA group;the serum mass concentrations of OVA-specific IgG2a in the HP-β-CD+Q group were about 1.3 times as high as those in the ISCOMs and Quil A groups and about 6.5 times as high as those in the OVA group.The splenic lymphocyte stimulation index was significantly higher in the HP-β-CD+Q and ISCOMs groups than that in the OVA group(P<0.05).The number of splenic CD4^(+)lymphocyte subpopulations was extremely significantly lower in the HP-β-CD+Q and ISCOMs groups than that in the OVA group(P<0.01),and significantly lower in the Quil A group than that in the OVA group(P<0.05).The number of splenic CD19^(+)lymphocytes in the ISCOMs group was significantly lower than that in the OVA group(P<0.05).The results indicated that HP-β-CD could enhance the adjuvant activity of Quil A,stimulate the body to produce high levels of antibodies,and possibly mediate the enhanced antibody response by promoting the migration of CD4^(+)to peripheral lymphoid tissues.