【作者】 张红英；

【导师】 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2007， 博士

【摘要】 猪蓝耳病即猪繁殖与呼吸综合征（porcine reproductive and respiratorysyndrome,PRRS）是由猪蓝耳病毒（猪繁殖与呼吸综合征病毒,porcinereproductive and respiratory syndrome virus,PRRSV）引起的一种以妊娠母猪流产、各种年龄猪（特别是仔猪）呼吸障碍为特征的高度传染性疾病。PRRSV感染导致猪免疫功能低下,对其他致病因子的易感性增加。目前PRRS几乎遍及世界各养猪国家,给世界养猪业造成了巨大的经济损失。对该病尚无有效的治疗方法,临床上主要使用PRRSV疫苗进行免疫预防。PRRSV弱毒苗免疫效果较好,但本身存在散毒危险,灭活苗虽然安全性较高,但由于引起的体液免疫应答不完全,导致免疫效率不高,且PRRSV的抗体依赖性增强作用（ADE）,可能会导致染毒猪长期持续性感染。因此提高疫苗免疫的保护性抗体水平和细胞免疫作用是防制PRRSV感染的关键。多糖具有增强免疫、抗病毒等功能。本文选用板蓝根多糖（IRPS）、黄芪多糖（APS）和山药多糖（CYPS）分别进行了体外、体内抗病毒作用及免疫试验;从细胞水平、分子水平研究了其对PRRSV的免疫调节作用,初步揭示了3种植物多糖免疫调节作用的机理,为PRRSV疫苗免疫增强剂的筛选提供了理论依据。1.采用现代的微波处理与传统的水煎醇沉相结合的方法从板蓝根、黄芪、山药中提取植物多糖,并用观察细胞病变（CPE）和中性红法研究了它们对PRRSV感染Marc-145细胞的影响。结果发现:板蓝根多糖、黄芪多糖和山药多糖的提取率(g·kg^-1)分别为59.1、37.4和53.6,糖含量（%）分别为56.7、65.8和73.5。APS对PRRSV感染具有显著的阻断作用(在0.391 mg·mL^-1～0.049 mg·mL^-1),在较高浓度时具有显著的直接杀灭作用（P＜0.05）。IRPS对PRRSV感染具有显著的阻断作用(在0.196 mg·mL^-1～0.024mg·mL^-1安全浓度范围内)和直接杀灭作用（在较高浓度,P＜0.05）。CYPS在高浓度区(0.196mg·mL^-1～0.098 mg·mL^-1)可以有效阻断PRRSV对细胞的感染作用,对PRRSV无直接杀灭作用。表明这3种植物多糖均可阻断PRRSV对Marc-145细胞的感染,但对侵入细胞后的PRRSV无抑制作用。2.通过巨噬细胞吞噬功能、溶血素及溶血空斑试验研究了不同剂量的3种多糖对小鼠免疫功能的影响,结果证实3种植物多糖均能增强小鼠巨噬细胞的吞噬功能,提高溶血空斑及溶血素水平。板蓝根多糖、黄芪多糖和山药多糖提高小鼠免疫功能的最佳剂量分别为20 mg·kg^-1、30 mg·kg^-1和50 mg·kg^-1。3.以板蓝根多糖、黄芪多糖和山药多糖作为免疫增强剂分别与PRRSV灭活苗、弱毒苗联合免疫仔猪,用ELISA方法检测免疫猪血清中抗PRRSV抗体水平,结果发现,同一种多糖对不同类型的疫苗免疫增强作用不同,其效果和剂量密切相关。板蓝根多糖高剂量对PRRSV灭活苗、弱毒苗均能提高免疫抗体水平;低剂量板蓝根多糖能提高PRRSV灭活苗、降低PRRSV弱毒苗免疫抗体水平。黄芪多糖能显著提高PRRSV灭活疫苗的免疫抗体水平,高剂量的效果较好;与PRRSV弱毒苗同时使用,能抑制PRRSV抗体产生,其抑制作用和剂量密切相关,剂量越大抑制作用越强,特别是在免疫的早期。山药多糖可显著提高PRRSV灭活苗免疫抗体水平,以低剂量效果较好,山药多糖与弱毒苗同时使用,会降低PRRSV抗体的水平。4.研究了板蓝根多糖、山药多糖和黄芪多糖对体外培养的猪脾脏淋巴细胞增殖、以及分泌一氧化氮（NO）、IL-2、IL-4和IFN-γ的影响。结果表明:这3种植物多糖体外均能促进猪淋巴细胞的增殖,其作用具有一定的量效关系。黄芪多糖、山药多糖均能显著促进猪脾淋巴细胞NO的合成;板蓝根多糖高剂量抑制NO的合成,低剂量促进NO的合成。3种多糖均可提高淋巴细胞分泌IFN-γ的量,其中板蓝根多糖效果最好;3种多糖均有促进淋巴细胞分泌IL-4的功效,且黄芪多糖效果最好;3种多糖对IL-2的分泌均有抑制作用。这从细胞水平、分子水平揭示了多糖免疫调节的机制。5.运用流式细胞技术检测了3种多糖对PRRSV疫苗免疫猪外周血T淋巴细胞亚群的影响,结果发现3种多糖和PRRSV灭活苗联合使用,均能促进猪CD3⁺细胞的增殖,其中以山药多糖和板蓝根多糖效果较好;3种多糖对CD4⁺细胞的增殖有一定的促进作用,其中以山药多糖低剂量组（CYPS_L）、山药多糖高剂量组（CYPS_H）和黄芪多糖高剂量组（APS_H）效果较好;3种多糖均能促进猪CD8⁺细胞的增殖,其中以板蓝根多糖和山药多糖效果较好。3种多糖分别和PRRS弱毒苗联合使用,对猪外周血CD3⁺细胞、CD4⁺细胞的影响不显著;免疫早期可促进猪外周血CD8⁺细胞的增殖,其中以板蓝根多糖和黄芪多糖效果较好。多糖和不同类型疫苗联合使用,对T淋巴细胞亚群的影响不同。6.用实时荧光定量PCR研究了3种多糖分别与PRRSV弱毒苗联合使用对猪外周血淋巴细胞中IFN-γmRNA表达的影响,结果发现:黄芪多糖和山药多糖与PRRSV弱毒苗联合使用在免疫的早期会抑制猪外周血淋巴细胞中IFN-γmRNA的表达,而板蓝根多糖对猪外周血淋巴细胞中IFN-γmRNA表达的影响和剂量密切相关,高剂量可以提高表达而低剂量则抑制表达。这从核酸分子水平揭示了多糖免疫调节的机制。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome virus （PRRSV） is the causative agent of porcine reproductive and respiratory syndrome （PRRS） firstly recognized in North America in 1987. PRRS is characterized by reproductive failures in late-term pregnant sows, high mortality in unweaned piglets and respiratory problems in pigs of all ages. PRRS has been a major health problem to swine herds worldwide and causes great economic loss in pork production industry since it first appeared. Many strategies aimed to control and eradicate PRRS have not always been effective, possibly due to the characteristic properties of the virus such as genetic and antigenic variations, subclinical and persistent infections, viremia with concurrent antibody, and antibody-dependant enhancement. Therefore, development of safer and more effective immunization remains a big challenge.Polysaccharides possess immunity enhancing and antiviral activities. In this study, three herbal polysaccharides were selected as immunoregulationer, their antiviral and immune-regulating abilities were investigated through a series of experiments in vitro and vivo. The purpose is to offer the theory basis for developing polysaccharides as immunopotentiators.1. Three herbal polysaccharides were extracted respectively from Astragalus, isatis root and Chinese yam through water-extraction and ethanol-precipitation. The total contents （%） of Astragalus polysaccharides （APS）, isatis root polysaccharides （IRPS）, and Chinese yam polysaccharides （CYPS） were 65.8, 56.7 and 73.5 respectively, measured by Vitriol-anthracene keton.. The total extractive rates (g·kg^-1) of them were 37.4, 59.1 and 53.6 respectively. To evaluate the antiviral activity and mechanism of the three polysaccharides, APS, IRPS and CYPS were respectively applied to Marc-145 cell culture before, after and simultaneous with PRRSV respectively. The cytopathic effect （CPE） were observed and the OD values of the cultures were determined by neutral red colorant absorption method to evaluate the effects of three polysaccharides against virus infection. The results showed that three Chinese herbal polysaccharides could decrease CPE lesions and inhibit virus from infecting the cells in a dosage dependent mode when the polysaccharides were added early to PRRSV, APS and IRPS also displayed antiviral activity when simultaneously added with PRRSV, but the three polysaccharides had no effect on the virus in the infectural cells.2. Effects of the three polysaccharides on immunity of mice were studied by testing the intraperitoneal macrophages function, the formation of haemolytic plaque and seral hemolysin. The results showed that the three herbal polysaccharides could improve the function of intraperitoneal macrophages, raise the level of haemolytic empty spots and seral hemolysin, and the optimal effective dosages were respectively 30mg·kg^-1 of APS , 20mg·kg^-1 of IRPS and 50mg·kg^-1 of CYPS.3. In order to study the effects of the three polysaccharides on specific-antibody levels in response to PRRS inactivated or live virus vaccine, piglets were inoculated with vaccines along with APS, IRPS and CYPS at high or low dose respectively. The results showed that: one polysaccharides extract had different effects on immuno-regulation when applied with different types of vaccine, and the potential were closely correlated to its dose. IRPS at high dosage could enhance the antibody titers induced by inactivated and live PRRS virus vaccine, whereas it just enhanced the antibody titer induced by inactivated vaccine but decreased that by live virus vaccine at low dosage .APS could enhance the antibody titer for inactivated vaccine, the high dose being more potent, but it showed opposite effects on live virus vaccine, the higher dose, the lower titer. CYPS could enhance the antibody titer for inactivated vaccine prominently, the low dose being better, but decreased the titer for live virus vaccine.4. Effects of the three polysaccharides in vitro on proliferation of swine splenic lymphocytes and secretion of cytokines and NO were studied. The results showed that at suitable dosages the three herbal polysaccharide could stimulate proliferation of splenic lymphocyte induced by ConA or LPS significantly （p<0.01） in vitro, and effects of three polysaccharides were related to their dosage. APS and CYPS could stimulate excretion of NO at any dose, and CYPS effected more strongly. IRPS could improve excretion of NO at low dose but inhibit at high dose. The three polysaccharide could promote the production of IL-4 （p<0.01） , and APS was the best. Remarkable upregulation of IFN-γco-induced by ConA and polysaccharide were observed in all three polysaccharides, and IRPS was the best , CYPS being the next. The boosting effection of polysaccharides on IFN-γand IL-4 was related to their dose. In contrast, the three polysaccharides appeared to inhibit the secretion of IL-2 significantly （p<0.05）5. Effects of the three polysaccharides on T subpopulations in the immune response to PRRS inactivated or live virus vaccine were studied. The results showed that: in the inactivated vaccine co-inoculating groups, the three polysaccharides could improve the proportion of CD3⁺ and CD8⁺ in pheripheral blood of piglets, CYPS and IRPS being better than APS. In the live vaccine co-inoculating groups, the three polysaccharides improved the proportion of CD8⁺ at the early stage, IRPS and APS being better than CYPS, but all had no influence on CD3⁺ and CD4⁺. It indicated that the effect of the herbal polysaccharides on T subpopulations in the immune response to PRRS were related to the type of vaccine. The results would reveal the immuno-regulation of polysaccharides at cellular level in vivo.6. Effects of the three polysaccharides on IFN-γmRNA expression in the immune response to PRRS live virus vaccine were studied by real-time fluoresence quantitative PCR （RT-FQ-PCR）. The results showed that APS and CYPS could down-regulate the IFN-γmRNA expression in porcine PBMC, especially at the early stage, while IRPS affected the IFN-γmRNA expression dose-dependently. High dose IRPS promoted IFN-γmRNA expression and low dose depressed it. It would reveal the immuno-regulation of polysaccharides at nucleic acid level in vivo.更多还原