【作者】 孙建华；

【导师】 胡松华；

【作者基本信息】 浙江大学 ， 预防兽医学， 2007， 博士

【摘要】 本文研究了八种不同结构达玛烷型人参皂甙(Ginsenosides) Rg3、Rd、Rc、Rb1、Rb2、Rg1、Re和Rg2的免疫佐剂作用,初步探讨不同类型、不同化学结构人参皂甙与其佐剂活性强弱之间的关系。对试验使用的人参皂甙的溶血性进行了检测以考察其用于局部注射的安全性。进一步探讨八种人参皂甙单体中佐剂作用较强的Rg1与氢氧化铝(Aluminum hydroxide, Alum)联合应用的佐剂效果。本文还在细胞水平探讨人参皂甙的佐剂作用机理。1、达玛烷型人参皂甙的溶血作用体外溶血试验结果表明,Rb1在浓度为1 mg/ml时未出现溶血性现象。Rg1有微弱的溶血现象出现,在1 mg/ml的浓度时,溶血百分比为5.28%,而QA溶血性相对较大,在8μg/ml的浓度时即已达到100%溶血。提示,本研究中使用的人参皂甙作为佐剂局部注射比较安全。2、八种不同结构达玛烷型人参皂甙佐剂作用的研究以卵清白蛋白(Ovalbumin, OVA)为模式抗原,研究了二醇组人参皂甙(Protopanaxadiol-type) Rg3、Rd、Rc、Rb1和Rb2,以及三醇组(Protopanaxatriol-type) Rg1、Re和Rg2的佐剂活性。55只BALB/c被随机分成11组,在第1,21天分别腹部皮下注射生理盐水、卵清白蛋白(OVA)、OVA+Rg3、OVA+Rd、OVA+Rb1、OVA+Rc、OVA+Rb2、OVA+Rg1、OVA+Re、OVA+Rg2或OVA+QA。每只小鼠OVA的注射量为10μg,各种皂甙的注射量均为50μg。二免后2周,采血分离血清用于检测OVA特异性IgG总抗体及抗体亚类IgG1和IgG2a水平。同时分离脾脏用于淋巴细胞增殖试验和细胞因子的检测。结果显示,Rg1、Re、Rg2、Rg3和Rb1可显著增强OVA特异性抗体的产生(P＜0.05)。而Rd、Rc和Rb2未达到显著水平(P＞0.05)。Rg1显著增强了脾淋巴细胞体外受丝裂原刀豆蛋白A (Concanavalin A, Con A)、脂多糖(Lipopolysaccharide, LPS)和抗原OVA刺激的增殖反应(P＜0.05),以及受OVA刺激产生Interleukin-5 (IL-5)和Interferon-γ(IFN-γ)的水平(P＜0.05),推测Rg1、Re、Rg2、Rg3和Rb1可能是人参皂甙中主要的佐剂活性成分。三醇组人参皂甙佐剂作用较强,二醇组人参皂甙佐剂作用微弱或几乎无佐剂作用,但Rg3除外。进一步分析和比较8种人参皂甙的佐剂活性和结构的关系发现,各种人参皂甙佐剂活性的差异可能与结合在达玛烷型骨架上的糖侧链所决定的分子构象的不同有关。3、Rg1与氢氧化铝联合佐剂效果研究研究了Rg1及Rgl+Alum对OVA诱导的Th1和Th2类免疫应答的作用。结果显示,Rg1对OVA特异性IgG(P<0.05或P<0.01)、IgGl (P<0.05或P＜0.01)和IgG2a (P<0.01或P＜0.001)抗体水平,IFN-γ和IL-4水平(P＜0.05)及慢性迟发型变态反应(Delayed-type hypersensitivity, DTH) (P<0.01)均具有显著的提高作用。Rgl(50μg)+Alum (200μg)与Alum (200μg)相比,显著增强了OVA特异性IgG抗体、DTH反应和细胞因子IFN-γ和IL-5的水平(P＜0.05或P＜0.01),尤其是Th1类反应,包括IgG2a抗体水平(P＜0.01)、脚掌肿胀系数(P＜0.05)和IFN-γ水平(P＜0.01)。结果提示Rg1单独或Rg1+Alum可以用作佐剂来对于Th1/Th2类免疫应答均有显著促进作用。4、Rg1佐剂作用机理的探讨实时定量PCR (Real-time PCR, RT-PCR)分析体外Rg1对小鼠巨噬细胞(Macrophages, Mφ)的Toll样受体(Toll-like receptor, TLR 4)和诱导型一氧化氮合成酶(inducible nitric oxide synthase, iNOS) mRNA水平的影响。结果表明,Rg1与M(?)体外共培养12 h后,可以显著提高腹腔M(?)的TLR 4 (P<0.01)和iNOS (P <0.05) mRNA的表达。结果提示,Rg1很可能通过提高TLR mRNA的表达来激活天然免疫系统和发挥免疫调节作用。更多还原

【Abstract】 Adjuvant effects and structure-effect relations, as well as the haemolytic activities of eight dammarane type saponins from Panax ginseng were evaluated. Meanwhile, the adjuvant effect of Rgl+Alum was estimated, and the adjuvant mechanism of Rgl was investigated. The above results provided a basis for development and application of medicinal herbs in vaccine adjuvants.1. Evaluation of haemolytic activity of dammarane-type ginsenosidesThe haemolytic activities of Rbl and Rgl were detected, and compared with that of QA. Results demonstrated that Rb1 showed no haemolytic activity and Rg1 showed slight haemolytic effect (P< 0.05) with 5.28% haemolysis at the concentration of lmg/ml. However,100% haemolysis was observed at concentration of 8μg/ml of QA. The results indicated that ginsenosides could be safe for use as adjuvants.2. Adjuvant effects and structure-effect relations of dammarane-type saponins from ginseng rootsThe objective of this test was to characterize the adjuvant effects of protopanaxadiol saponins (Rg3, Rd, Rc, Rb1 and Rb2) and protopanaxatriol saponins (Rg1, Re and Rg2) isolated from the root of Panax ginseng C. A. Meyer. Fifty-five BALB/c mice were subcutaneously injected twice at a three-week interval with 0.89% saline or 0.89% saline containing 10μg of ovalbumin (OVA) or 0.89% saline containing 10μg of OVA plus 50μg of ginsenosides Rg3, Rd, Rc, Rb1, Rb2, Rg1, Re or Rg2 or Quil A. Two weeks after the boost, blood samples were collected for measuring specific total-IgG, IgG1 and IgG2a responses, and splenocytes were harvested for the detection of lymphocyte proliferation as well as Interferon-γ(IFN-γ) and Interleukin-5 (IL-5) production. The results indicated that OVA-specific antibody responses were significantly higher (P< 0.05) in mice immunized with OVA co-administered with Rg1, Re, Rg2, Rg3 and Rb1 but not with Rd, Rc and Rb2 (P> 0.05) when compared with the control (immunized with OVA alone). Significantly enhanced splenocyte proliferative responses to Concanavalin A (Con A), Lipopolysaccharide (LPS) and OVA (P<0.05) as well as the production of both IL-5 and IFN-y (P<0.05) stimulated by OVA were detected in mice immunized with OVA co-administered with Rg1 but not with Rb1, Re and Rg3 when compared with the control. Therefore, among the ginsenosides studied, Rg1, Re, Rg2, Rg3 and Rb1 are more potent adjuvant than the others, indicating that they are the major constituents contributing to the adjuvant activities of total ginseng saponins. Variety of ginsenosides in adjuvant activity might be attributed to the variety of molecular conformations determined by the side sugar chains attaching to their dammarane skeleton.3. Ginsenoside Rgl combined with Alum promoted a balanced Thl and Th2 immune responses to ovalbumin in mice.In present work, the adjuvant potential of Rgl and Rgl+Alum on Thl and Th2 immune responses of BALB/c mice against ovalbumin (OVA) were evaluated. Results demonstrated that Rgl showed adjuvant effects on Thl/Th2 immune responses by enhancing both IgGl (P< 0.05 or P< 0.01) and IgG2a (P< 0.01 or P< 0.001) levels, as well as DTH (delayed-type hypersensiticity) reaction (P< 0.01). Furthermore, the antigen-induced cytokine secretions (IFN-γand IL-5) in splenocyte culture were significantly enhanced by Rgl compared with OVA control (P< 0.05). Interestingly, Rg1 (50μg) combined with Alum (200μg) showed significant increase in OVA-specific antibody responses, DTH reaction and cytokine secretions (IFN-y and IL-5). In conclusion, results suggest that Rgl alone or plus Alum could be used as a safe adjuvant via up-regulating both Th1- and Th2-like immune responses.4. Possible mechanisms of adjuvant activity of RglThe aim of this study was to evaluate the effects of Rgl on mRNA levels of Toll-like receptor 4 (TLR 4) and inducible nitric oxide synthase (iNOS) in mouse peritoneal Mcp. The results showed that Rgl significantly increased the mRNA levels of TLR 4 (P< 0.01) and iNOS (P< 0.05) of Mcp treated with Rgl for 12 h. It is probably that Rgl could activte the innate immune system and showed immune modulate effects by raising the mRNA level of TLR gene.更多还原