【作者】 柳风祥；

【导师】 崔治中；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2009， 博士

【摘要】 畜禽疾病特别是病毒性疾病仍然是阻碍养殖业健康发展的瓶颈,疫苗是防治疾病最有效的方法。但是,近几年由于免疫抑制性病毒的普遍存在,预防工作变得异常艰难,国内外学者一直致力于研究提高疫苗的特异性和非特异性免疫增强作用。中药是我国传统文化的结晶,是中华民族的文化瑰宝,在畜禽疾病的防治过程中起过重要的作用。中药免疫增强剂对机体的细胞免疫和体液免疫功能具有增强或调节作用,可提高机体的抗病力和抵抗力,显示了显著增强机体免疫的作用。本研究以临床上常常发生的网状内皮组织增生症病毒(REV)感染雏鸡,造成一定程度的免疫抑制,建立试验动物模型,比较中药提取物对不同免疫状态下鸡的免疫调节作用,并对主要成分多糖进行提取分离鉴定和分子量测定,为下一步多糖的结构和功能研究奠定基础。1中药提取物免疫调节作用的研究1.1中药提取物和盐酸左旋咪唑对鸡的免疫调节作用分别在临床健康鸡和在1日龄感染网状内皮增生症病毒(REV)诱发了免疫抑制的鸡测试了中药提取物和盐酸左旋咪唑对鸡的免疫调节作用,以及对鸡体重、免疫器官的影响。结果表明,在饮水中添加50ppm的盐酸左旋咪唑,无论在健康鸡还是免疫抑制鸡对NDV、H5-AIV灭活疫苗免疫后的HI抗体水平、体重和免疫器官指数都没有显著影响(p>0.05)。由黄芪、党参、淫羊藿和甘草组成的提取物饲喂健康鸡对NDV和H5-AIV免疫后的HI抗体水平也没有显著影响(p>0.05)。但是在REV诱发了免疫抑制的鸡,相对于不喂中药提取物的对照组,该中药提取物可显著提高对NDV、H5-AIV的HI抗体水平和免疫器官指数(p<0.01),虽然仍显著低于未经REV感染的对照鸡。该研究表明,由黄芪、党参、淫羊藿和甘草组成的提取物对REV诱发的免疫抑制状态下的鸡有显著的免疫增强作用,盐酸左旋咪唑没有免疫增强作用。1.2不同组合中药提取物对鸡的免疫调节作用分别在临床健康鸡和在1日龄感染网状内皮增生症病毒(REV)诱发了免疫抑制的鸡测试了不同浓度黄芪提取物和由黄芪、党参、淫羊藿、甘草组成的不同组合的中药提取物对鸡的体液免疫调节作用,以及对鸡体重、免疫器官的影响。结果表明,不同浓度的单一黄芪提取物对NDV和H5-AIV免疫后的HI抗体滴度没有显著影响,对鸡的体重、免疫器官指数也没有显著影响(P>0.05)。由黄芪、党参、淫羊藿和甘草混合物组成的中药提取物,在健康鸡对NDV和H5-AIV灭活疫苗免疫后的HI抗体滴度没有显著影响。但是在REV诱发了免疫抑制的鸡,该混合中药提取物可显著提高对NDV和H5-AIV的HI抗体滴度(p<0.01),虽然仍显著低于未经REV感染的对照鸡。该中药制剂还可以显著增加胸腺和法氏囊指数,即缓和由REV诱发的萎缩。该研究表明,单一黄芪不能提高由REV诱发的免疫抑制鸡的抗体水平,黄芪、党参、淫羊藿和甘草组成的中药提取物对REV诱发的免疫抑制鸡具有显著的免疫增强作用。13不同提取方法中药提取物对鸡的免疫调节作用分别在临床健康鸡和1日龄感染网状内皮组织增生症病毒(REV)诱发了免疫抑制的鸡测试了经不同提取方法获得的中药提取物的免疫调节作用,并研究水溶性提取物对不同来源鸡的免疫调节作用。结果表明,在饮水中添加由黄芪、党参、淫羊藿和甘草组成的提取物,无论是水溶性提取物(水提取物和乙醚提取后的水提取物)还是脂溶性提取物(乙醚提取物),对健康鸡NDV和H5-AIV灭活疫苗免疫后HI抗体水平均无显著影响(P>0.05),水溶性提取物对REV诱发的免疫抑制鸡的NDV和H5-AIV灭活疫苗免疫后HI抗体水平有显著影响(p<0.01),可显著提高其抗体水平。但是,由同样药物组成的脂溶性提取物却没有免疫增强作用(P>0.05)。中药提取物对健康鸡和SPF来源继续在SPF下饲养的鸡对NDV和H5-AIV灭活疫苗免疫后的HI抗体滴度没有显著影响(p>0.05)。但是在REV诱发了免疫抑制的鸡和SPF来源在非SPF条件下饲养的鸡,该中药提取物可显著提高对NDV和H5-AIV的HI抗体滴度(p<0.01),虽然仍显著低于未经REV感染的对照鸡。1.4不同分子量段中药提取物对鸡的免疫调节作用用网状内皮增生症病毒(REV)感染海蓝褐公鸡和SPF来源的鸡诱发免疫抑制,在饮水中添加中药提取物或不同分子量段的中药提取物,测试其对鸡的免疫调节作用。结果表明,同样由黄芪、党参、淫羊藿和甘草组成的提取物和分子量段在1-10万道尔顿的提取物,不论是先混合、提取再超滤、浓缩还是先分别提取、超滤再浓缩、混合可显著提高感染REV鸡的NDV和H5-AIV的HI抗体滴度(p<0.01),虽然仍显著低于未经感染REV的对照鸡。但分子量小于1万道尔顿和大于10万道尔顿的提取物,对感染REV的免疫抑制鸡没有显著免疫增强作用(p>0.05)。试验结果还表明,中药提取物和分子量在1-10万道尔顿的提取物对SPF来源的鸡在非SPF环境下饲养有显著的免疫增强作用(p<0.05),对同样是SPF来源的鸡继续在SPF环境下饲养却没有显著的免疫增强作用(p>0.05)。该研究表明,由黄芪、党参、淫羊藿和甘草组成的提取物其免疫增强作用的分子量在1-10万道尔顿之间,而对SPF来源继续在SPF环境下饲养的鸡没有显著免疫增强作用。1.5中药提取物对鸡外周淋巴细胞转化率的影响为了检测中药提取物对外周淋巴细胞转化率的影响,采用中药提取物对感染网状内皮组织增生症病毒(REV)鸡外周血液、胸腺和脾脏淋巴细胞转化率进行试验,建立MTT法来观察淋巴细胞的转化率。结果显示,REV显著抑制雏鸡血液、胸腺和脾脏淋巴细胞转化率(p<0.05)；中药提取物对健康雏鸡血液、胸腺和脾脏淋巴细胞转化率没有显著的影响(p>0.05),可显著提高由REV诱发的免疫抑制鸡35日龄前血液、胸腺淋巴细胞转化率和21日龄前脾脏淋巴细胞的转化率(p<0.05),对49日龄后血液、胸腺和35日龄后脾脏淋巴细胞转化率影响不明显(p>0.05)。2多糖的提取分离鉴定和分子量测定2.1中药提取物化学成分初步鉴定本试验用中药提取物化学成分的鉴定方法鉴定了由黄芪、党参、淫羊藿和甘草组成的用不同方法提取的混合物的成分。结果表明,用水浸提后得到的提取物含有鞣质、多糖及其苷类、皂苷、氨基酸、多肽和蛋白质,用乙醚浸提后得到的提取物含有生物碱、黄酮及其苷类、挥发油、油脂类,用乙醚浸提后再用水浸提得到的提取物和用水浸提后得到的提取物成分相同,水浸提后的提取物用超滤膜过滤后,分子量小于1万道尔顿的提取物含有鞣酸、氨基酸、多肽和蛋白质,分子量在1-10万道尔顿的提取物中含有多糖及其苷类、皂苷、多肽和蛋白质,分子量大于10万道尔顿的提取物含有糖、多糖及其苷类、皂苷、多肽和蛋白质。水溶性成分和脂溶性成分的区别在于水溶性成分含有糖、多糖、皂苷,而脂溶性成分没有,证明多糖及其苷类和皂苷具有免疫增强作用。2.2中药粗多糖的提取参数研究本试验采用四因素三水平正交设计研究了提取温度(A),料液比(B),提取时间(C)及提取次数(D)对中药粗多糖得率的影响。最佳提取条件参数为A3B1C3D3,即浸提温度80℃,料液比1：10,浸提时间3h,提取次数3次。采用苯酚-硫酸法测得复方中药多糖提取物GCPS中多糖含量为48.45%,测试结果稳定可靠。苯酚-硫酸法是目前测定多糖含量较可靠的传统方法之一,本法简单快速,灵敏度高,重现性好。2.3中药多糖的分离纯化和分子量测定由黄芪、党参、淫羊藿和甘草组成的复方中药提取物,按Sevag法脱除蛋白质后得复方粗多糖GCPS。粗多糖分别用终浓度50%、60%和70%的乙醇沉淀,离心,干燥,得到三种多糖组分(GCPS-1、GCPS-2和GCPS-3);将分级醇沉所得多糖组分分别进行DEAE-纤维素柱层析,得三种主要多糖GCPS-1A, GCPS-2A, GCPS-3A,分别进行TOYOPEARL HW-55F凝胶柱层析,得三种主要成分GCPS-1AⅠ, GCPS-2AⅡ, GCPS-3AⅢ。经过Sephadex G-200凝胶柱层析、紫外可见分光光度分析和纸层析鉴定,表明是均一组分,不含有核酸和蛋白质杂质；并测得均一组份GCPS-1AⅠ中的多糖含量为77.6%,GCPS-2AⅡ中的多糖含量为95.2%,GCPS-3AⅢ中的多糖含量为87.3%；根据已知分子量的标准葡聚糖Dextran系列的洗脱体积得到标准曲线方程：Y=-0.1734X+0.8676,R=0.9993,并根据标准方程测得GCPS-1AⅠ, GCPS-2AⅡ及GCPS-3AⅢ的相对分子质量(Mr)为301254、94541和12453。3结语本研究首次应用REV作为免疫抑制因素建立试验动物模型,研究了由黄芪、党参、淫羊藿和甘草组成的提取物对不同免疫状态下雏鸡和SPF鸡的免疫调节作用,不同分子量段提取物的免疫调节作用,不同提取方法提取物的免疫调节作用,不同组合中药提取物的免疫调节作用,并与盐酸左旋咪唑进行了比较。还进一步研究了中药提取物对试验动物细胞免疫的调节作用。建立了应用REV作为测试中药提取物免疫增强作用的试验模型,证实了复方中药提取物的免疫增强作用。而且这种有效成分是水溶性的,分子量在1-10万道尔顿之间。但具体的化学成分还有待于进一步研究。本研究还对起主要免疫增强作用的化学成分多糖进行提取、分离和鉴定,探索了多糖的最佳提取条件,测试了均一多糖的含量和分子量。更多还原

【Abstract】 Poultry diseases, especially viral diseases, remain an obstacle to the development of animal husbandry. Vaccine is the most effective way to prevent disease. However, because of immune suppressive diseases prevalence in recent years, prevention has become exceptionally difficult. Scholars have been working to improve the immune-enhancing role of vaccine. Herbal medicine is the crystallization of Chinese traditional culture and the cultural treasures of the Chinese nation. Herbal medicine have played an important role in the process of livestock and poultry diseases prevention. The immunoenhancer of herbal medicine enhance or adjust the body cellular and humoral immunity function and enhance the body’s resistance to disease, indicating a significant role in enhancing immune. reticuloendotheliosis virus (REV) infected in chickens usually in clinical practice, and resulted in a certain degree of immune suppression. In present study, animal model was established, and to compare the immunoregulatory of herbal extractions in the different immune status of chickens. The polysaccharide as a major components were isolated, identificated and determinate the molecular weight, the structure and function will be found in further study.1 Immune regulation of herbal extraction1.1 Effectiveness of herbal extraction and levamisole hydrochloride on immune regulation in chickensThe immune regulation of herbal extraction and levamisole hydrochloride was investigated in health chickens and immune suppressive chickens infected with REV in 1-day-old. The results showed that levamisole hydrochloride added 50mg/kg in water did not effect hemagglutination inhibition (HI) antibody titers both in the health chicken and the immune suppressive chickens after immunization with Newcastle disease virus (NDV) and H5 subtype of avian influenza virus (H5-AIV) vaccine(p>0.05), the same results was showed in body weight and the relative weight of immune organs. Herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae did not effect HI antibody titers in the health chicken after immunization with NDV and H5-AIV vaccine (p>0.05), but it can significantly improve the HI antibody titers, body weight and the relative weight of immune organs in the immunosuppression chickens infected with REV(p<0.01), although the datas were still lower than the health chickens. The study showed that herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae had immune enhancement effects in chickens immnunosuppressed by REV, and levamisole hydrochloride no effects.1.2 Effectiveness of different combinations herbal extraction on immune regulation of chickensThe immune regulation of herbal extraction of different concentraction Radix Astragali and different combination composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae was investigated in health chickens and immune suppressive chickens infected with REV in 1-day-old. The results showed that herbal extraction of different concentraction Radix Astragali was not effect on the NDV and H5-AIV of HI antibody titers in different immune status chickens, and not effect on the body weight and the relative weight of immune organs as well(p>0.05). The immune regulation of herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae did not effect HI antibody titers in the health chicken immuned with NDV and H5-AIV vaccine(p>0.05), but it can significantly improve the HI antibody titers in the immunosuppression chickens infected with REV(p<0.01), although the datas were still lower than the health chickens. The herbal extraction could also increase the relative weight of immune organs. The study showed that a single Radix Astragali could not increase HI antibody titers in immunosuppressive chickens infected with REV. The herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae had immune-enhancement in immunosuppressive chickens infected with REV.1.3 Effectiveness of herbal extraction extracted by different methods on immune regulation in chickensThe immune regulation of herbal extraction extracted by different methods was investigated in health chickens and immune suppressive chickens infected with REV in 1-day-old and that of water-soluble herbal extraction was researched in different origin chickens. The results showed that the herbal extraction composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae was added in water, no matter the herbal extraction was water-soluble extraction (water extraction and water extraction after extracted with ethyl ether) or fat-soluble extraction (ethyl ether extraction), did not effect HI antibody titers in health chicken after immuned with NDV and H5-AIV vaccine (p>0.05). The water-soluble extraction could increase the HI antibody titers of the chickens infected with REV, but the fat-soluble extraction the same composition had not immune-enhancement (P>0.05). The water-soluble extraction did not effect HI antibody titers both in the health chicken and the SPF chickens feeded in FPF environment (p>0.05), but it can significantly improve the HI antibody titers in the immunosuppressive chickens and SPF chickens feeded in non-SPF environment infected with REV(p<0.01), although the datas were still significantly lower than the health chickens.1.4 Effectiveness of herbal extraction of different molecular weights on immune regulation in chickensThe immune regulation of herbal extraction and different molecular weght of herbal extraction was investigated in different immune status chickens and SPF chickens infected with REV in 1-day-old. The results showed that the herbal extraction and the 1-10 million Dalton molecular weight of herbal extraction composed of Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae, no matter the herbal extraction was mixed firstly, extracted, ultrafiltered and concentrated or extracted, ultrafiltered, concentrated respectively and then mixed, can significantly improve the H5-AIV and NDV of HI antibody titers in immunosuppressive chickens induced with REV(p<0.01), although the datas were still lower than the control without REV-infected chickens. However, less than 10,000 Dalton and greater than 100,000 Dalton molecular weight of the herbal extraction had not immune-enhancement in immunosuppressive chickens infected with REV (p>0.05). This results also showed that the herbal extraction and the 1-10 million Dalton molecular weight of herbal extraction of had immune-enhancement in immunosuppressive SPF chickens infected with REV feeded in non-SPF environment (p<0.05), but had not immune-enhancement in the same chickens feeded in SPF environment(p>0.05). The research showed that the molecular weight of the herbal extraction with immune-enhancement composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae was between 1-10 million Dalton, and had not immunoenhancement in the SPF chickens feeded in SPF environment.1.5 Effectiveness of herbal extraction on peripheral lymphocyte proliferation in chickensThe study was conducted to detect the effects of herbal extraction on peripheral blood, thymus and spleen lymphocytes proliferation in chicken infected with REV. MTT method was used to observe lymphatic cell proliferation. The results showed that the blood, thymus and spleen lymphocyte proliferation were immune-suppressived in chickens infected with REV (p<0.05). Herbal extraction had no effects on the lymphocytes proliferation of blood, thymus and spleen in health chicken (p>0.05), but it can increased that of the blood, thymus before 35-day-old and the spleen before 21-day-old in chickens infected with REV(p<0.05), which has not effects the lymphocyte proliferation of blood, thymus after the 49-day-old and the spleen after 35-day-old in chickens(p> 0.05).2 Extracting, isolating, identifing and determining of molecular weight of polysaccharide2.1 The chemical constituents identification of herbal extractionThe mixture components of herbal extraction composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae was identified by the different motheds of chemical constituents identification. The results showed that herbal medicine was distilled by water contains tannin, sugar and their glycosides, saponins, amino acids, peptides and proteins, was extracted with ethyl ether contains alkaloids, flavonoids and their glycosides, volatile oil, fat class. The herbal medicine was distilled by water after extracted with ethyl ether firstly and was distilled by water was the same composition, after filtered with ultrafiltration membrane, the molecular weight of less than 10,000 Dalton of the herbal extraction contains tannic acid, amino acids, peptides and proteins,1-10 million Dalton contains sugars, polysaccharides, glycosides, saponins, peptides and proteins, more than 100,000 Dalton contains sugar, polysaccharides and their glycosides, saponins, peptides and proteins. The difference of fat-soluble components and water-soluble components of herbal extraction is that water-soluble components contain sugar, polysaccharides, saponins and fat-soluble components do not contain that. polysaccharides and saponins and their glycosides have a role in immune-enhancing.2.2 The study of extracting parameters of herbal extraction polysaccharideThe effect was investigated by a four-factor three-level orthogonal design of the extraction temperature (A), ratio of herb to liquid (B), extraction time (C) and extracting times (D)of on the yield of crude polysaccharide. The best extract parameters was A3B1C3D3, that the extraction temperature was 80℃, ratio of herba to liquid was 1:10, extraction time was 3h, and extract times were 3 times. The compound polysaccharide content of GCPS was 48.45% measured by phenol-sulfuric acid method. The test results were stable and reliable. Phenol-sulfuric acid method was more reliable determination of the polysaccharide content of the traditional methods. This method was simple, rapid, high sensitivity and good reproducibility.2.3 The study of purification and molecular weight identification of herbal extraction polysaccharidesThe compound polysaccharide(GCPS) was obtained from the herbal extraction composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae removaled of protein by Sevag method. The three polysaccharide (GCPS-1, GCPS-2 and GCPS-3) was obtained from GCPS by precipitation, centrifugation and drying using ethanol of final concentration 50%,60% and 70%. The three major polysaccharide(GCPS-1A, GCPS-2A, GCPS-3A) derived from above polysaccharide(GCPS-1, GCPS-2 and GCPS-3) was obtained with DEAE-cellulose column chromatography respectively, which were chromatographyed with TOYOPEARL HW-55F gel column and then getting the three major components of a GCPS-1A I, GCPS-2A II, GCPS-3AⅢ. After identified by Sephadex G-200 gel column chromatography, UV spectrophotometry and paper chromatography the composition (GCPS-1, GCPS-2 and GCPS-3) was homogeneous, does not contain nucleic acid and protein impurities. The polysaccharide contents of homogeneous composition (GCPS-1, GCPS-2 and GCPS-3) was 77.6%,95.2% and 87.3% respectively. The standard curve equation is obtained from series elution volume of known molecular weight dextran Dextran:Y=-0.1734X+0.8676, R=0.9993, and measured the molecular weigh(Mr) of GCPS-1AI, GCPS-2AII and GCPS-3AIII according to the standard equation:301254, 94541 and 12453. 3 InclusionThe experimental animal model was established firstly by REV as immunosuppressive factors to study the immune enhancement of herbal extraction composed with Radix Astragali, Radix Codonopis, Herba Epimedii and Radix Glycyrrizae in different immune status chickens or SPF chicken, different molecular weight extraction, extraction with different methods, different combinations of extraction and compared with levamisole hydrochloride. Further the herbal extraction was used to research the regulation of cell-mediated immunity in experimental animals. The experimental animal model was established by REV as immunosuppressive factors to determinate the immune enhancement of herbal extraction, confirme the immune enhancement of compound herbal extraction. And the active components of the herbal extraction were water-soluble and that molecular weight is between 1-10 million Dalton. However, the specific chemical composition has yet to be studied in the future.The polysaccharide as a major components of immune enhancement was isolated, identificated and determinated the molecular weight and content of uniformity polysaccharides for further study of the structure and function. The best extraction conditions of polysaccharide is investigated also.更多还原