【作者】 储岳峰；

【导师】 李祥瑞；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2003， 硕士

【摘要】 从6种常用传统中草药中提取制备了9种免疫增强活性成分，应用免疫学技术，以从中筛选作为新型免疫增强剂的成分或配方，并探索其作用机理。采用体内实验与体外实验相结合的观察方式，探讨了他们各自对小鼠特异性免疫因素、非特异性免疫因素、免疫细胞和免疫分子的作用效应，并通过动物实验比较了它们各自在临床应用时的增强免疫效果，最终筛选出了一部分中药活性成分，作为低毒、高效的新型免疫增强剂配方。 实验1，为本研究提取和制备了9种中药活性成分，分别为：当归多糖(77.28％)、黄芪多糖(82.48％)、板蓝根多糖(82.94％)、淫羊藿多糖(66.38％)、蜂胶多糖(51.41％)、蜂胶黄酮(46.92％)、淫羊藿黄酮(38.37％)、黄芪黄酮(1.39％)、黄芪皂甙(72.88％)。 实验2，采用腹腔注射的给药方式，测定了9种中药成分对正常小鼠脾脏和外周血淋巴细胞增殖反应的影响。结果表明，9种药物的促进ConA和LPS诱导小鼠外周血和脾脏淋巴细胞增殖的活性，可以排序为淫羊藿多糖＞黄芪多糖，蜂胶黄酮＞人参皂甙，当归多糖＞蜂胶多糖，板蓝根多糖＞淫羊藿黄酮＞黄芪皂甙，同时根据各药物的作用特点筛选出了体内实验的合适剂量，分别为多糖类20mg／Kg·d，黄酮和皂甙类5mg／Kg·d。 实验3，观察了9种中药成分对体外培养的小鼠淋巴细胞增殖的影响。按照刺激的强弱可以排序为淫羊藿多糖、黄芪多糖、人参皂甙、蜂胶黄酮＞淫羊藿黄酮＞当归多糖＞黄芪皂甙，与体内实验相似，揭示体内体外的实验结果有较强的相关性；而且根据各药物体外作用的特点选择了合适的体外实验用剂量，多糖类为20ug／ml，黄酮和皂甙类为5ug／ml。 实验4，应用半定量RT-PCR方法，测定了9种中药成分对小鼠脾脏T细胞IL-2mRNA水平的影响。按照T细胞内IL-2mRNA水平的高低，可将9种成分的作用效应排序为：黄芪多糖＞淫羊藿多糖＞蜂胶黄酮＞当归多糖＞黄芪皂甙＞蜂胶多糖＞板兰根多糖＞人参皂甙＞淫羊藿黄酮。 实验5，将9种中药成分按体内实验确定的剂量给小鼠腹腔注射后，分别测定其腹腔巨噬细胞功能和脾脏NK细胞杀伤活性的变化。按腹腔巨噬细胞的活性强弱排序为人参皂甙＞黄芪多糖＞蜂胶黄酮＞淫羊藿黄酮＞当归多糖＞淫羊藿多糖＞蜂胶多糖，按NKC杀伤率大小排序为人参皂甙＞蜂胶黄酮＞黄芪多糖＞淫羊藿黄酮＞淫羊藿多糖＞蜂胶多糖＞当归多糖，其余成分作用不显著。 实验6，用绵羊红细胞定量溶血测定法代替空斑溶血实验，检测了9种中药成分对小鼠脾脏抗体分泌细胞数量的影响。结果显示，按照光吸收值大小可排列为人参皂戒＞蜂胶黄酮＞黄茂多糖＞淫羊察黄酮＞淫羊落多糖＞黄艺皂么，其余成分则低于对照。 实验7，将合适剂量下的9种中药成分和（或）LPS加入到体外培养的小鼠脾脏淋巴细胞体系中，共培养5天后测定培养物上清里的 IgG水平。结果发现按照0D490值的大小可以排列为：人参皂武＞蜂胶黄酮、黄艺多糖＞淫羊茶多糖＞当归多糖＞淫羊霍黄酮，其余成分低于对照。 实验8，9种中药成分在兔瘟组织灭活疫苗免疫家兔前连续3天分别皮下注射，用微量血凝抑制实验（HI）进行体液免疫水平测定，观察各成分对兔瘟组织灭活疫苗的免疫增强效果。结果发现，在最大杭体效价上比较各成分的增强作用，则可排序为：人参皂忒＞淫羊茶多糖＞蜂胶黄酮＞黄艺多糖＞当归多糖＞淫单薄黄酮＞其他成分。 根据这些实验的结果我们发现，各活性成分的作用机理与作用特点不尽相同，我们可脚它们的免疫活性强弱，将黄民多糖、人参皂么、淫羊茗多糖、蜂胶黄酮作为新型免疫增强剂的首选对象，将当归多糖、淫羊渡黄酮、黄艺皂洲为备选。更多还原

【Abstract】 In order to screen the novel immunostimulant from Chinese Herbal Medicine ingredients (CHMIs), we extracted nine bio-active ingredients from five kinds of CHMs, and investigated on their mechanism with the technology of immunity. Effects of the nine ingredients on the specific and non-specific factors in immunology, immunocytes and immuno-molecular in vivo and in vitro were studied, and their immno-enhancing activity in clinic were compared. Then several ingredients of these CHMIs were selected as the novel immunostimulant.Section 1. Nine bio-active ingredients were extracted from five kinds of CHMs, including astragaluspolysaccharide (APS), propolis polysaccharide (PPS), epimedium polysaccharide (EPS), Chinese angelica polysaccharide (CAPS), isatis root polysaccharide(IRPS), astragalus flavone(AF), propolis flavone(PF), epimedium flavone(EF), astragaloside ( AS). And net content (%) of every ingredients are 77. 28, 82.48, 82.94, 66.38, 51.41, 46.92, 38.37, 1.39, 72.88 respectively.Section 2. To detect the immune activities and the target cells of the CHMIs, the stimulating activities of these CHMIs on peripheral blood and splenic lymphocytes in mice were compared. All CHMIs could be arranged as EPS, APS, PF, GS, CAPS, PPS, IRPS, EF, AS, according to which they stimulated lymphocytes to proliferate in vivo. In this study, we also selected the best stimulating dose in vivo of every CHMI by the difference of their stimulating activities, and there are 20mg/Kg .d in polysaccharides, 5 mg/Kg.d in f lavones, Ginsenoside and Astrogaloside.Section 3. The stimulating activities of nine CHMIs on peripheral blood and spleenic lymphocytes cultured in vitro were compared. All CHMIs could be made a turn about EPS, APS, GS, PF, EF, CAPS, AS, according to which they stimulated lymphocytes to proliferate in vitro, and which is similar to that of in vivo. In this study, we also selected the best stimulating dose in vitro of every CHMIs by the difference of their stimulating activity, and there are 20ug/ml in polysaccharides, 5 ug/ml in flavones, Ginsenoside and Astrogaloside.Section 4. In this study, the IL-2mRNA levels in normal mice T cells stimulated by nine CHMIs was determined using a reverse transcription and semi-quantification polymerase chain reaction. The results showed that all CHMIs would be arranged in turn by the level of IL-2mRNA they stimulated like this: APS>EPS>PF>CAPS>AS>PPS>IRPS>GS>BF.Section 5. The acticity of natural killer(NK) cell cytotoxic and murine peritoneal macrophages(PM) in mice were determined after the mice were injected the nine CHMIs, respectively. The results showed that APS, EPS, CAPS, PF, EF and GS enhanced PM activity significantly, furthermore, the six and PPS improved NK cells cytotoxic activity markedly.Section 6. 50 mice were divided into 10 groups and immunized APS, EPS, PPS, CAPS, IRPS, PF, EF, AS and GS, respectively, then the production of spleen ASC were observed for all groups 4 days after immunization by SRBC, in the way of quantitative haemolysis of SRBC. It was found that all CHMIs could be made in a turn of GS>PF>APS>EF>EPS>AS.Section 7. Nine CHMIs in the proper dose and (or) LPS added into spleen lymphocytes cultured in vitro, and the IgG levels were measured 5 days after with sandwich ELISA. In this study, all CHMIs would be arranged in turn by the level of IgG they stimulated like this: GS>APS, PF>EPS>CAPS>EF.Section 8. Healthy young rabbits were injected the nine CHMIs three times in 3 days before they were immunized with the vaccine of RVHD, respectively, and HI employed to determine the level of antibody immunity. It was found that they would be arranged like GS, EPS, APS, PF, CAPS and EF according to their enhancing activity.All of these results suggest that these CHMIs have different stimulating activities and different target cells in mice. APS, GS, EPS, PF were screened clinically for the animal immune enhancers, and CAPS, EF, AS second.更多还原