【作者】 李科；

【导师】 黄庆洲；

【作者基本信息】 西南大学 ， 基础兽医学， 2008， 硕士

【摘要】 肉苁蓉多糖（Polysaccharide of Cistanche deserticola,CDPS）是从植物肉苁蓉中提取的一类高分子活性物质,其可增强巨噬细胞吞噬功能、提高血清溶血素值,同时还具有单独或协同ConA、PHA促进小鼠胸腺淋巴细胞增殖、脾脏淋巴细胞分泌IL-2的能力,显示出广泛的免疫调节功能。但将CDPS作为一种免疫增强剂直接用于兽医临床,存在着体内代谢较快、作用时间较短、使用成本较高等不足之处。脂质体作为一种人工合成的药物靶向载体,对所载药物具有缓释、靶向沉积于网状内皮系统（RES）的特性,能延长药物的作用时间、提高生物利用效率及药物定向作用于免疫系统的强度。本课题利用脂质体包封技术,将CDPS制备成一种新型的免疫增强剂即肉苁蓉多糖脂质体,通过均匀试验设计原理,筛选出制备CDPS脂质体的最佳工艺参数,并以目前仍严重危害养鸡业发展的病毒性传染病鸡新城疫（NewcastleDisease,ND）为研究对象,探讨CDPS脂质体对ND疫苗免疫效果的影响,并从细胞免疫、体液免疫、免疫调节因子（IL-2、IL-4、IFN-γ）活性变化方面,研究CDPS脂质体增强肉鸡免疫机能的可能机制。主要研究内容及结果如下:1.三种方法提取肉苁蓉多糖的比较研究分别采用水提醇沉法、超声法、超声结合水提法提取肉苁蓉多糖,胰蛋白酶-Sevag法除去蛋白,利用molish鉴别反应、碘-碘化钾反应、多糖水解反应鉴定提取物是否主要为多糖类物质,葸酮-硫酸比色法测定提取物中多糖的含量。结果三种方法的提取物主要均为多糖,其得率分别为1.93%、2.28%、6.06%,纯度分别为65.7%、74.3%、81.2%,说明超声结合水提法提取肉苁蓉多糖效果最好。2.均匀试验设计法优化肉苁蓉多糖脂质体制备工艺研究采用逆向蒸发法制备CDPS脂质体,透析处理后利用蒽酮-硫酸比色法测定包封率,并以其为CDPS脂质体质量的主要评价指标。根据均匀试验设计原理,按照U*₆（6⁴）均匀设计表设计试验方案,探讨大豆卵磷脂与氯仿的比例、胆固醇与大豆卵磷脂的比例、肉苁蓉多糖与大豆卵磷脂的比例、磷酸盐缓冲液PH值4个因素（每个因素6水平）对包封率的影响。结果,四个因素分别为7%、1∶5、1∶14、6.6时,CDPS脂质体平均包封率最高,达68.13%,RSD值仅为2.51%,且在此条件下制备的CDPS脂质体在4℃、避光保存120天内,PH值基本稳定,虽有分层现象出现,但保存120天时包封率仍达50.15%。3.肉苁蓉多糖脂质体对鸡ND苗免疫效果的影响研究选择20日龄健康罗曼肉公鸡60只随机分为4组,即CDPS脂质体（Ⅰ）、CDPS（Ⅱ）、脂质体（Ⅲ）和生理盐水（Ⅳ）组,当试验鸡血清HI抗体效价低于3log2时肌肉注射免疫ND苗,同时,Ⅰ组肌肉注射5mg/ml的CDPS脂质体（500μl/只）、Ⅱ组肌肉注射50mg/ml的CDPS（200μl/只,CDPS含量为Ⅰ组的4倍）、Ⅲ组肌肉注射空白脂质体（500μl/只）、Ⅳ组肌肉注射生理盐水（500μl/只）。分别于免疫前及免疫后第10、20、30天时,翅静脉采血收集血清,测定其HI抗体效价及IgG含量,同时对免疫后30天时所收集血清进行鸡胚中和保护试验。结果免疫后10天时,试验组及对照组HI抗体效价明显升高;免疫后20天时,Ⅰ组极显著高于生理盐水对照组（P＜0.01）,略高于Ⅱ、Ⅲ组;免疫后30天时,四组血清HI抗体效价均呈降低趋势,但Ⅰ组显著高于Ⅱ组和Ⅳ组（P＜0.05）,与空白脂质体组无显著差异（P＞0.05）;免疫鸡血清IgG含量变化趋势显示:免疫后10天时,Ⅰ组IgG水平较其它组略高,但各组间无显著差异（P＞0.05）,Ⅰ组免疫后第20、30天,Ⅱ、Ⅲ组免疫后第30天时,血清IgG水平均极显著地高于对照组（P＜0.01）,免疫后30天时,Ⅰ组还极显著高于CDPS、空白脂质体组（P＜0.01）,Ⅰ组免疫后10天至30天时,血清IgG水平呈逐渐升高趋势,其它组免疫后10天至20天时,血清IgG水平呈升高趋势,但至30天时,血清IgG水平开始下降;中和试验结果显示:CDPS脂质体组鸡胚死亡率明显低于CDPS、空白脂质体及生理盐水组,中和指数以CDPS脂质体组最高。结果说明,CDPS脂质体对肉鸡免疫机能的促进作用最强。4.肉苁蓉多糖脂质体对鸡免疫ND苗后血液中T淋巴细胞、IL、IFN活性的影响研究在对肉鸡进行ND苗抗体检测的同时,于免疫后15天,每组随机取6羽,翅静脉无菌采集抗凝血,采用酸性α-乙酸萘酯酶染色试验（ANAE）和淋巴细胞转化试验（MTT法）分别测定T淋巴细胞ANAE阳性率及增殖活性。免疫后20天时,每组随机取8羽翅静脉采血分离血清,采用ELISA方法测定血清中细胞因子IL-2、IL-4、IFN-γ的含量。结果表明,淋巴细胞转化率在四组间无显著差异（P＞0.05）,淋巴细胞ANAE阳性率在Ⅰ、Ⅱ、Ⅲ组间无显著差异（P＞0.05）,但Ⅰ、Ⅱ组显著高于对照组（P＜0.05）;IL-2含量以CDPS脂质体组和脂质体组较高,均极显著高于CDPS组和生理盐水对照组（P＜0.01）,CDPS脂质体组IL-4含量极显著高于其它三组（P＜0.01）,IFN-γ的含量在Ⅰ、Ⅱ、Ⅲ组间无显著差异（P＞0.05）,但CDPS脂质体组显著高于生理盐水组（P＜0.05）。以上结果说明:肉苁蓉多糖脂质体对肉鸡免疫机能的促进作用与血清中T淋巴细胞、IFN-γ、IL-4、IL-2的活性变化相关。更多还原

【Abstract】 Polysaccharide of cistanche deserticola（CDPS）,extracted from cistanche deserticola,is a kind of active substance with high molecular.The CDPS can enhance the phagocytosis ability of macrophage,increase the value of erythrocytolysin in serum;at the same time,it can facilitate the proliferation of thymic lymphocyte,secretion of IL-2 of lymphocyte from spleen in mice alone and with ConA,PHA together,showed an extensive immune regulating function.If taking CDPS as a immunoenhancer applied to animal directly,there will be some deficients,includ quick metabolism, short action time and high cost.As a type of synthetic targeting vector,slow-release and targeting to endothelial system（RES）are significant characteristics of liposome,it can prolong action time of drugs,and enhance bioavailable efficiency and the intension which drugs orient effect on immune systcm.Based on all above mentioned,the purpose of this study is to prepare the CDPS liposome,as a new type of immunoenhancer,the parameter for preparation of CDPS liposome was selected with homogeneous design.Then the effect of CDPS liposome on chickens after immunization with ND Lasota vaccine was exarned.The function of cellular immunity,humoral immunity and the activity exchange of IL-2、IL-4、IFN-γhave been studied,in order to stduy the possible mechanisms of how CDPS liposome enhance immunologic function.The main contents arc as follows:1.Comparation of three methods to extract polysaccharide from cistanche deserticolaPolysaccharide of cistanche deserticola（CDPS）was extracted with three methods,including hot water,supersound,hot water-supersound and precipitation by alcohol.Protein in the precipitates was removed by Sevag-trypsin.CDPS was identified by molish reaction,iodine-potassium iodide reaction,colorimetry with Fehling’s reagent.Polysaccharide in extractive was determined by the anthrone-sulphuric acid colorimetry.Content of pick-up rate was 1.93%,2.28%,6.06%respectively, and the content of polysaccharides in extractive was 65.7%,74.3%,81.2%respectively.The results indicated that hot water-supersound was the most adequate method to extract polysaccharide in cistanche deserticola.2.Study on optimum parameter of preparation of CDPS liposme with homogeneous designThe CDPS liposme was prepared by the reverse-phase evaporation method.Entrapment efficiency,as main index of quantitive evaluation of liposome,was determined by dialysis and anthrone-sulphuric acid colorimetry.Based on principle of homogeneous design and table U^*₆（6⁴）, the program of experiment was designed,Four factors,including the ratio of soybean lecithin and chloroform,cholesterol and soybean lecithin,CDPS and soybean lecithin,PH of buffer phosphate on entrapment efficiency was investigated.The results showed the entrapment efficiency was 68.13% and RSD was 2.51%when the four factors were 7%,1:5,1:14,6.6 respectively.In these conditions the PH of CDPS liposome was stable on day 120^thand the entrapment efficiency was 50.15%when preserved at 4℃away from light although the stratification was observed.3.Study on effect of CDPS liposome on La sota vaccine immunized chickenSixty chickens with similar health condition were regarded as the experimental animals,which were randomly divided into 4 groups,with 15 chickens in each group at 20 day-old.Experimental chickens were injected with ND Lasota vaccine when the antibody titer of HI was under 31og2.ThenⅠgroup were injected with CDPS liposome（concentration:5mg/ml,dose:500μl for one bird）,Ⅱgroup were administered with CDPS（concentration:50mg/ml,dose:250μl for one bird）,Ⅲgroup were administered with liposome（500μl for one bird）,Ⅳgroup were injected with saline（dose: 500μl for one bird）.Levels of IgG（indirect ELISA）,HI titer and serum neutralization test were detected at different times The result of HI suggested that the antibody titer of all groups had risen obviously on the 10^thday after immunization,compared with preimmune.On the 20^thday after immuned,compared with control group,the antibody titer ofⅠgroup had risen obviously（P＜0.01）, it was also slightly higher thanⅡ,Ⅲgroup.Antibody titer of four groups descented continuously on 30^thday,but the antibody titer ofⅠgroup was higher thanⅡ,Ⅳgroup（P＜0.05）,at the same day,there were no significant deviation betweenⅠgroup andⅢgroup.The result of indirect ELISA suggested that IgG levels ofⅠgroup was slightly higher than other groups on 10^thday after immunization,,it was significantly higher than control group on 20^th,30^thday（P＜0.01）,it was also significantly higher thanⅡ,Ⅲgroup on 30^thday,（P＜0.01）.IgG levels ofⅠgroup had risen continuously on 30^thdays,IgG levels of other groups had risen from 10^thday to 20^thday,but had decreased from 20^thday to 30^thday.The result of Serum neutralization test suggested that mortality of chick embryo inⅠgroup was lower thanⅡ,Ⅲ,Ⅳgroup.Neutralization index ofⅠgroup kept the highest in all groups.These results indicated that CDPS liposome,as a new type of immunoenhancer,could boost effective immune response after immunization with ND Lasota vaccine.4.Study on effect of CDPS liposome on activity of T lymphocyte,IL,IFN of La sota vaccine immunized chickenAfter injected ND La sota vaccine,6 chickens,which were randomly obtained from each group were bled and sera from them were detected by MTT assay and acidα-naphthyl acetate esterase test respectly for diterminatoin of lymphocyte transformationt ratio and ANAE⁺ ratio on 15^thday after immunization.On 20^thday,8 immunized chickens were bled and sera from them were tested by ELISA for the determination of the levels of IL-2,IL-4,IFN-γ.These results suggested that lymphocyte proliferation of four groups were no significantly deviation,and ANAE⁺ ratio of lymphocyte of groupⅠ,groupⅡ,groupⅢwere no significantly deviation,and ANAE⁺ ratio ofⅣ lymphocyte of groupsⅠandⅡwere higher than the control group（P＜0.05）,the IL-2 level of groupsⅠandⅢhad risen obviously,compared withⅡandⅣgroups;the IL-4 level of CDPS liposome group kept the highest in all groups,the content of IFN-γwas no significantly deviation amongest groupsⅠ,ⅡandⅢ;but the IFN-γlevel of CDPS liposome group was higher than the control group,these results indicated there were correlation between effect of CDPS liposome on effective immune response of chicken and activity of T lymphocyte,IL-2,IL-4,IFN-γ.更多还原