【作者】 廉宜君；

【导师】 谷新利；

【作者基本信息】 石河子大学 ， 临床兽医学， 2007， 硕士

【摘要】 多糖的非特异性免疫调节作用给近代免疫缺损疾病、肿瘤及艾滋病的治疗开辟了新方向。作为免疫增强剂,低毒、无残留的中药复方多糖的研究开发将具有广阔的市场前景和良好的社会意义。本文对G-中药复方免疫增强剂中多糖的提取参数、分离纯化方法及免疫调节作用进行了研究,论文主要内容如下:首先以粉碎脱脂的G-中药复方为原料,研究了浸提温度、料液比、浸提时间和提取次数对热水浸提法提取中药复方多糖得率的影响,通过正交实验确定了水提法提取中药复方多糖的最佳提取参数,即浸提温度60℃,料液比1:15 ,浸提时间4h,提取次数3次。在上述提取条件下,水提法提取中药复方多糖得率可达到12.65%。采用多重分析法将中药复方多糖与黄芪多糖、复方水煎剂对正常小鼠的免疫作用做比较,结果显示,中药复方多糖的免疫兴奋作用最佳,显著优于复方水煎剂和黄芪多糖(P<0.05),而复方水煎剂效果略优于黄芪多糖,提示中药复方多糖是免疫增强剂提高机体免疫功能的主效成分。在脱蛋白工艺研究中,比较了Sevag法、木瓜蛋白酶法与木瓜蛋白酶﹢Sevag法三种脱蛋白方法的效果。蛋白质去除率分别为53.7%、43.9%、70.8%;多糖损失率分别为35.8%、3.2%和12.3%。从蛋白去除和多糖提取两方面综合考虑,确定木瓜蛋白酶与Sevag法结合作用为除蛋白最佳工艺。采用乙醇分级沉淀、DEAE-纤维素柱层析和TOYOPEARL HW-55F凝胶柱层析分离得到了纯白色的GCPS-1AⅠ,GCPS-2AⅡ及GCPS-3AⅢ,经过Sephadex G-200凝胶柱层析法、紫外分光光度法及纸色谱纯度鉴定说明GCPS-1AⅠ,GCPS-2AⅡ及GCPS-3AⅢ为均一组分。采用TOYOPEARL HW-55F凝胶柱层析法测得三种多糖均一组分的相对分子质量为:GCPS-1AⅠ,398107;GCPS-2AⅡ,122138 ;GCPS-3AⅢ,7951。考察中药复方多糖均一组分GCPS-1AⅠ,GCPS-2AⅡ及GCPS-3AⅢ对Cy造成的免疫抑制模型小鼠的免疫调节作用,发现多糖均一组分GCPS-2AⅡ可使环磷酰胺诱导的免疫抑制小鼠的免疫脏器器官恢复至接近正常水平;恢复单核巨噬细胞系统的吞噬功能,提示对机体非特异性免疫有增强作用;使小鼠迟发型超敏反应能力恢复并略超过正常水平,提示对T淋巴细胞介导的细胞免疫有增强作用;使经过绵羊红细胞抗原刺激后小鼠的抗绵羊红细胞抗体产生能力增强,提示对免疫功能低下的小鼠的体液免疫有增强作用。GCPS-1AⅠ和GCPS-3AⅢ对抗Cy所致的免疫抑制效果不明显。更多还原

【Abstract】 Polysaccharides bring new hope for therapying immune-defected disease,tumour,AIDS through the way of non-specific antigen.As compound immunoenhancer,low-toxic and non- rudimental compound polysaccharides will have good market.In this paper, polysaccharides from compound immunoenhancer were extracted and separated and researched the effects on immunity function.The suitable extraction techniques for water-soluble compound polysaccharides from extrusion defatted traditional chinese medical herbs were established.The factors that affected the extraction of compound polysaccharides included the ratio of material to solvent,extracting time, temperature and times of extracting.Aorthogonal experiment was designed .the result showed that the optimal conditions were at the temperature of 60℃, extracting time 4 hour, the ratio of material to solvent1:15 and times of extracting 3.The efficiency of extracting polysaccharides from compound immunoenhancer would be 12.65% at these conditionFifty mice were randomly divided into five groups, Chinese herbal medicine compound polysaccharide group, Chinese medical formula decoction group, astragalus polysaccharide group, normal saline group. A series of immunity tests are carried out. Conclusion: the Chinese herbal medicine compound polysaccharide can obviously increase the immunity function in mice. Chinese medical formula decoction groupis better than astragalus polysaccharide group.During the removing protein process, Sevag method, papain method and Sevage method with papain method together have been compared. The protein scavengin- g rate was 53.7%, 43.9%, 70.8 %; the polysa ccharides losing rate was 35.8%, 3.2%, 12.3 %; The Sevag method with papain method together had not only more polysaccharides extraction percent, but also the highest remov- ing protein percent , so it was theoptimal removing protein process.Three polysaccharides GCPS-1AⅠ, GCPS-2AⅡand GCPS-3AⅢ,were got using ethanol–depositing, DEAE-Cellulose ion-exchange chromatography and TOYOPEARL HW-55F gel chromatography. Three polysaccharides were proved homogeneous by Sephadex G-200 gel chromatography, paper chromatography and ultraviolet spectrophotometer. The relative molecular weights of three homogeneous polysaccharides were determined 398107, 122138, and 7951 by TOYOPEARL HW - 55F gel chromatography.GCPS-2AⅡcan stimulate humoral,cellular and non-specific immunity by oral administration in mice with immunosuppressive agent Cyclophophamide(Cy).JP. injection of Cy can inhibit the proliferation of splennocytes and the genesis of dilated type hypersensitivity,and enhance humoral immunity such as antibody response. GCPS-1AⅠand GCPS-3AⅢcan’t reduce immune suppressant caused by Cy.更多还原