【作者】 张萍；

【导师】 江善祥；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2015， 博士

【摘要】 畜禽传染性疾病,尤其是病毒性疾病,是危害畜牧业发展的首要问题,由于它们能造成畜禽养殖业的巨大损失而在全世界范围内备受关注。因病毒具有独特的生物特征和发病机理,目前还没有针对病毒性疾病有效的治疗方法,所以疫苗接种则成了主要的预防措施。虽然有关传染性疾病疫苗的研究和应用很多,但现有疫苗的免疫效果却不尽人意,很多疫苗由于病毒的易变异性和理化因素等影响,经常出现免疫失败的现象。近年来,在畜禽免疫方面,中药多糖增强免疫应用受到了兽医界医药科研者的广泛关注。研究证明,许多中药及其组分具有一定的抗病毒和增强免疫作用,一些中药及其组分在疫苗接前后使用,这样可以提高机体免疫力,降低传染病发病率。灵芝(Ganoderma lucidum)在我国应用历史悠久,灵芝的药用成分非常丰富,尤其是从灵芝中提取的灵芝多糖(Ganoderma lucidum polysaccharides,GLP)。近年来试验研究表明,灵芝多糖具有增强免疫功能、抗病毒、抗炎症、抑制肿瘤、降血压、护肝等作用,上述研究主要集中在人医上,且相关作用已得到多方面的实验验证,但灵芝多糖在畜禽生产方面的研究还很有限,尤其是在畜禽生产中抗病毒和增强免疫作用方面的研究甚少。因此,本论文以赤灵芝多糖为研究目标,通过相关体内外试验,研究了赤灵芝多糖抗病毒与免疫调节作用及其机理,为赤灵芝多糖在养禽养殖上的应用以及在此基础上进一步开发新型免疫增强剂奠定基础。具体试验安排分以下八个部分:1.赤灵芝多糖对NDV、IBDV感染细胞能力的影响目的:探究赤灵芝多糖的体外抗病毒活性作用。方法:将赤灵芝多糖与IBDV、NDV两种病毒分别以3种加样方式(先加药,先加病毒,病毒和多糖感作后同时加入)加入培养24 h的鸡胚成纤维细胞(CEF)上,MTT法测定细胞活性以评价赤灵芝多糖对鸡传染性法氏囊病毒(IBDV)、鸡新城疫病毒(NDV)两种病毒感染CEF能力的影响。结果:先加赤灵芝多糖后接种病毒的方式,赤灵芝多糖在9.766～625μg.mL-1时,其A570均显著大于病毒对照组;先加病毒后,赤灵芝多糖对NDV无明显抑制作用,且只有在高浓度才体现抑制IBDV作用;病毒和多糖感作后同时加入,赤灵芝多糖在9.766～625 μg·mL-1时,其A570均显著大于IBDV对照组,而所有的多糖组A570均显著大于NDV对照组。结论:赤灵芝多糖对IBDV、NDV两种病毒感染CEF有一定的抑制作用,尤其是提前给多糖方式效果较好,提示赤灵芝多糖对病毒吸附宿主细胞有阻断和抑制作用。2.赤灵芝多糖对CEF细胞中IFN-β的mRNA表达影响目的:探讨赤灵芝多糖对CEF抗病毒感染的能力的影响机理。方法:选用Oligo7软件设计了一对干扰素-β(Interferonβ,IFN-β)引物,用荧光定量PCR方法测定了赤灵芝多糖对鸡胚成纤维细胞IFN-β的mRNA表达的影响。结果:赤灵芝多糖能诱导CEF中IFN-β的表达,使IFN-β的mRNA转录水平升高,尤其是灵芝多糖在156.25μg·mL-1 时,能够显著促进 CEF 中 IFN-β 的表达(P<0.05),在 39.065 和 78.125μg·mL-1时能促进IFN-β mRNA的表达,但与对照组差异不显著。结论:结合之前抗病毒试验结果分析,赤灵芝多糖对CEF抗病毒作用的影响与其诱导产生的IFN-β有关,可能是IFN-β干扰了病毒吸附宿主细胞,这也许是赤灵芝多糖增强CEF抗病毒感染能力的分子机制之一。3.赤灵芝多糖对鸡外周血淋巴细胞、脾脏淋巴细胞增殖的影响目的:探讨赤灵芝多糖对体外培养鸡外周血淋巴细胞、脾脏淋巴细胞增殖的影响。方法:将不同浓度的赤灵芝多糖单独或协同伴刀豆素球蛋白(ConA)刺激仔鸡外周血淋巴细胞、脾脏淋巴细胞,采用MTT法测定淋巴细胞增殖的变化。结果:单独刺激外周血淋巴细胞时,GLP在0.070～2.25 mg·mL-1的剂量范围内,其吸光度值均显著大于细胞对照组(P<0.05),协同ConA刺激外周血淋巴细胞时,GLP+ConA组在0.141～2.25 mg·mL-1吸光度值显著大于ConA对照组(P<0.05)。单独刺激脾脏淋巴细胞时,GLP在0.035～2.25 mg·mL-1浓度时的吸光度值显著大于细胞对照组(P<0.05),协同ConA刺激脾脏淋巴细胞时,GLP+ConA组在0.035～2.25 mg·mL-1吸光度值显著大于ConA对照组(P<0.05)。结论:赤灵芝多糖在体外不仅能单独刺激鸡淋巴细胞增殖,与ConA具有协同刺激作用,且在一定范围内呈现明显的量效关系。4.赤灵芝多糖体外对鸡外周血淋巴细胞中IL-6、IFN-γ mRNA表达的影响目的:探究赤灵芝多糖增强免疫的机理。方法:用Oligo7软件设计了白介素-6(Interleukin 6,IL-6)引物、干扰素-γ(Interferonγ,IFN-y)引物,利用多糖辅助 ConA刺激的鸡外周血淋巴细胞,并从中提取总RNA。荧光定量PCR方法测定赤灵芝多糖对鸡外周血淋巴细胞IL-6、IFN-y的mRNA表达的影响。结果:赤灵芝多糖在合适的浓度下能够协同ConA促进外周血淋巴细胞中IL-6、IFN-y mRNA的表达,与ConA对照组相比,GLP在562.5μg·mL-1、281.25 μg·mL-1时能够显著促进IL-6 mRNA的表达(P<0.05),GLP 在 140.63 μg·mL-1 时能够显著促进 IFN-y mRNA 的表达(P<0.05)。结论:赤灵芝多糖在合适的浓度下能够促进IL-6、IFN-γ mRNA的表达,这可能是其增强免疫的机理之一。5.赤灵芝多糖对雏鸡黏膜免疫功能的影响目的:探讨赤灵芝多糖对免疫雏鸡黏膜免疫功能的影响。方法:1日龄非免疫罗曼蛋公鸡饲养至14日龄,随机选取225只,分为5组,除空白对照组外均用新城疫Ⅳ苗滴鼻、点眼免疫,1羽份/羽,28日龄二免。在每次免疫的同时,3个药物剂量组分别灌服不同浓度的赤灵芝多糖溶液,连续3天,免疫对照组和空白对照组不给药。在二免后第14天分别各组随机抽取5羽,放血致死,采取十二指肠和盲肠扁桃体,用免疫组化法测定肠绒毛和盲肠扁桃体SIgA阳性细胞数量。结果:赤灵芝多糖在50、25、12.5 mg·kg-1剂量时可以显著提高十二指肠和盲肠扁桃体的SIgA细胞阳性数,其中25 mg·kg-1剂量组SIgA水平最高。结论:赤灵芝多糖可以有效地提高SIgA阳性细胞数目,从而增强机体黏膜免疫。6.赤灵芝多糖对免疫雏鸡外周血淋巴细胞IL-6、IFN-γ mRNA表达的影响目的:探讨赤灵芝多糖对免疫雏鸡白介素-6(Interleukin 6,IL-6)、干扰素-γ(Interferonγ,IFN-γ)表达的影响,分析赤灵芝多糖体内增强免疫机理。方法:1日龄非免疫罗曼蛋公鸡饲养至14日龄,随机选取225只,分为5组,除空白对照组外均用新城疫Ⅳ苗滴鼻、点眼免疫,1羽份/羽,28日龄二免。在每次免疫的同时,3个药物剂量组分别灌服不同浓度的灵芝多糖溶液,连续3天,免疫对照组和空白对照组不给药。在首免后第28天分别各组随机抽取5羽,无菌心脏采血,分离淋巴细胞,并从中提取总RNA。用Oligo7软件设计IL-6引物、IFN-γ引物。荧光定量PCR方法测定赤灵芝多糖对免疫雏鸡外周血淋巴细胞IL-6、IFN-γ mRNA表达的影响。结果:赤灵芝多糖在50、25 mg·kg-1时能够显著促进IL-6、IFN-γ mRNA的表达(P<0.05)。结论:赤灵芝多糖在体内能够促进细胞因子IL-6、IFN-γ mRNA的表达,这可能是其增强免疫的机理之一。7.赤灵芝多糖对雏鸡新城疫疫苗免疫应答影响目的:研究赤灵芝多糖对新城疫免疫雏鸡外周血淋巴细胞增殖、免疫器官指数以及抗体效价的影响。方法:300只1日龄罗曼蛋公鸡饲养至14日龄,随机分为6组,除空白对照组外均用新城疫疫苗滴鼻、点眼免疫,1羽份/羽,28日龄时进行二免,在每次免疫的同时,分别灌服3种不同浓度的赤灵芝多糖溶液,药物对照组灌服黄芪多糖溶液,连续3天,免疫对照组和空白对照组不给药。分别在首免后的第7天、14天,21天、28天测定外周血淋巴细胞增殖活性、新城疫抗体效价,在首免后第28天随机抽取5只处死,称取胸腺、脾脏和法氏囊重量,计算免疫器官指数。结果:赤灵芝多糖在50、25 mg·kg-1时可以促进雏鸡外周血淋巴细胞增殖、提升机体血清抗体效价,提高免疫器官指数等。结论:赤灵芝多糖可以提高机体的免疫能力,增强疫苗的免疫效果。8.赤灵芝多糖对免疫雏鸡抗新城疫病毒影响目的:研究赤灵芝多糖对免疫雏鸡抗新城疫病毒影响。方法:280只1日龄罗曼蛋公鸡饲养至14日龄,随机分为7组,除空白对照组外均用新城疫疫苗滴鼻、点眼免疫,1羽份/羽,28日龄时进行二免,在每次免疫的同时,分别灌服3种不同浓度的赤灵芝多糖溶液,药物对照组灌服黄芪多糖溶液,连续3天,免疫对照组、病毒对照组和空白对照组不给药。在首免后的第28天,除空白对照组外,其他各组用新城疫病毒攻毒,记录各组发病状况,并统计各组的发病率、死亡率、保护率。结果:赤灵芝多糖组高、中剂量组鸡只的死亡率和发病率均显著低于病毒对照组(P<0.05),也低于APS对照组和免疫对照组。结论:赤灵芝多糖可以增强疫苗的免疫效果,提升机体的抗病毒能力。更多还原

【Abstract】 Animal infectious diseases,especially viral disease,were a major problem in livestoc k husbandry,which have caused huge losses and receive worldwide concern.Because of un ique biological features and pathogenesis of virus,there was no effective method for anti-vi rus therapy,so vaccination was the main preventive measure.Although with so many resear ch and application of vaccine,immunization effect was always unsatisfactory with phenom enon of immune failure.It has been demonstrated that many Chinese herbal medicines and their ingredients possess antiviral activity.Some Chinese herbal medicines and their’ingredients have been used in chickens after or before vaccination,resulting in the decrease of the incidence of infectious diseases and the increase in animal immunity.Ganoderma lucidum(G.lucidum)has been widely used as a remedy to promote health and longevity in Asia for thousands of years,especially G lucidum polysaccharides(GLP),which are water-soluble and extracted from G lucidum.Recent studies have shown that GLP has antioxidant activities,immunomodulating activities,anti-tumor activities and so on.However,there are few reports about the effects of GLP on animal infectious diseases especially the virosis,which cause great loss in the domestic animal and poultry industry.In this study,the antiviral and immune-enhancing action and mechanism of GLP were investigated through a series of experiments in vitro and vivo.The purpose of this research is to offer theoretical evidence for the development of a novel immunopotentiator in the domestic animal and poultry industry.The details are divided into eight parts as follows:Test 1 Effects of G.lucidum polysaccharide on cell infectivity of NDV and IBDVTo investigate the antiviral activity of Ganoderma lucidum polysaccharides(GLP)in vitro,GLP was added into chicken embryo fibroblasts(CEF)infected with avian infectious bursal disease virus(IBDV)and Newcastle disease virus(NDV)in three different mechanisms:pre-addition,post-addition,and mixed addition of polysaccharides with virus,respectively.The blank control group and the virus control group were included.The effects of GLP on cellular infectivity of virus were determined by MTT method.Results showed that in the pre-addition mode,the A570 values of GLP groups(9.766～625μg·mL-1)were significantly higher than that of the virus control group,in the post-addition mode,only the A570 value of GLP group at 625 μg·mL-1 was significantly higher than that of the IBDV control group,and other GLP groups did not have significant actions;in mixed addition of polysaccharides with virus mode,the A570 values of GLP groups at 78.125μg·mL-1 to 625 μg·mL-1 infected with IBDV were significantly higher than those of the IBDV control group,and the A570 values of all GLP groups infected with NDV were significantly higher than those of the NDV control group.The results indicated that GLP,if used properly,possesses potent antiviral effect on IBDV and NDV infection,particularly by pre-addition of polysaccharides,and it also indicated that GLP could prevent IBDV and NDV infection by blocking the virus adsorbed host cells.Test 2 Effect of G.lucidum polysaccharide on mRNA expression of IFN-P in CEFTo investigate the antiviral activity and mechanism of Ganoderma lucidum polysaccharides(GLP)in vitro,according to Primer Premier software with Oligo7,a pairs of RT-PCR primers of interferon-p were designed.Then,the effects of GLP on expression level of interferon-β mRNA was determined by semi-quantitative RT-PCR assay.The results showed that GLP could improve the expression level of interferon-β mRNA,especially at the dose of 156.25μg·mL-1,GLP significantly increased ConA-induced IFN-βmRNA expression(P<0.05).At 39.065 and 78.125 μg·mL-1 of GLP slightly,but not significantly,increased IFN-β mRNA expression in CEF.It suggested that GLP exerts its antiviral activity at least in part via promoting IFN-β gene expression.Test 3 Effects of G.lucidum polysaccharide on proliferation of chicken peripheral and splenic lymphocytesIn order to study the action of GLP on chicken peripheral lymphocytes proliferation in vitro,the proliferation was observed in peripheral and splenic lymphocytes treated with GLP.The lymphocytes proliferation was determined by MTT assay.Results showed that GLP at 0.070～2.25 mg mL-1 alone increased the A570 values in chicken peripheral blood lymphocytes in a concentration-dependent manner,at 0.141～2.25 mg·mL-1 GLP also enhanced ConA-induced A570 values(P<0.05).Besides,GLP at 0.035～2.25 mg·mL-1 alone increased the A570 values in chicken splenic lymphocytes(P<0.05),at 0.035～2.25 mg·mL-1 GLP also enhanced ConA-induced A570 values(P<0.05).It implied that GLP not only promoted cell proliferation of the lymphocytes,but also synergized with ConA in promoting proliferation of the lymphocytes.Test 4 Effects of G.lucidum polysaccharide on mRNA expression of interleukin-6 and interferon-y in chicken peripheral lymphocyte in vitroIn order to study the action and molecular mechanism of GLP in immunoenhancement,according to Primer Premier software with Oligo7,two pairs of RT-PCR primers of Interleukin 6(IL-6)and Interferon γ(IFN-γ)were designed.Then,the effects of GLP on expression level of IL-6 and IFN-γ mRNA were determined by semi-quantitative RT-PCR assay.The results showed that GLP at 562.5μg·mL-1、281.25 μg·mL-1 significantly enhanced ConA-induced the IL-6 mRNA expression(P<0.05),and at 140.63 μg·mL-1 significantly enhanced ConA-induced the IFN-y mRNA expression(P<0.05).This might be one of molecular mechanisms of GLP in immunoenhancement.Test 5 Effects of G lucidum polysaccharide on mucosal immunity in vaccinated chickensTo investigate the action and mechanism of GLP in mucosal immunity,225 14-day-old chickens were randomly assigned into equal five groups randomly.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in experimental groups were orally administrated respectively with the GLP at 3 concentrations,in vaccination control(VC)and BC group with physiological saline,once a day for three successive days.On days 14 after the second vaccination,five chickens were sampled randomly from each group for determination of secretory immunoglobulin A(SIgA)that is released into intestinal mucosa and cecal tonsil by immunohistochemistry method.The results showed that GLP at 50、25、12.5 mg·kg-1 could raise the content of SIgA and increase the number of SIgA positive cells,especially at 25 mg kg-1,the level of SIgA positive cells was the highest.It demonstrated that GLP could enhance the number of SIgA positive cells as so to enhance the immune function of mucosa,and improve the ability of anti-infection.Test 6 Effects of G.lucidum polysaccharide on mRNA expression of interleukin-6、interferon-y in vaccinated chickens peripheral lymphocyteTo evaluate the adjuvanticity and molecular mechanism of GLP in immunoenhancement in vaccinated chickens in vivo,225 14-day-old chickens were randomly assigned into equal five groups randomly.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in experimental groups were orally administrated respectively with the GLP at 3 concentrations,in vaccination control(VC)and BC group with physiological saline,once a day for three successive days.On days 28 after the first vaccination,five chickens were sampled randomly from each group for determination of the expression level of IL-6、IFN-γ mRNA by semi-quantitative RT-PCR assay.The results showed that GLP at 50 mg·kg-1、25 mg·kg-1 significantly enhanced the IL-6、IFN-γ mRNA expression(P<0.05),This might be one of molecular mechanisms of GLP in immunoenhancement.Test 7 Effects of G lucidum polysaccharide on immune response of Newcastle disease vaccine for chickensTo evaluate the adjuvanticity of GLP on immune response of Newcastle disease vaccine for chickens in vivo,300 14-day-old chickens were randomly assigned into equal six groups randomly.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in experimental groups were orally administrated respectively with the GLP at 3 concentrations,in drug control chickens were orally administrated respectively with Astragalus polysaccharide(APS),in vaccination control(VC)and BC group with physiological saline,once a day for three successive days.On Day 7,14,21 and 28 after the first vaccination,the peripheral lymphocytes proliferation,the serum ND antibody titer and the immune organ index were determined.The results showed that GLP at 50、25 mg·kg-1 could significantly promote lymphocyte proliferation,raise serum antibody titer,improve immune organ index of spleen,bursa and thymus.The results indicated that GLP may be a novel immunomodulator.Test 8 Effects of G lucidum polysaccharide on immune protective effects of Newcastle disease vaccine for chickensTo further study the antiviral activity of GLP on immune response of Newcastle disease vaccine for chickens in vivo,280 14-day-old chickens were randomly assigned into equal 7 groups randomly.The chickens except blank control(BC)group were vaccinated with Newcastle disease vaccine,repeated vaccination at 28 days old.At the same time of the first vaccination,the chickens in experimental groups were orally administrated respectively with the GLP at 3 concentrations,in drug control chickens were orally administrated respectively with Astragalus polysaccharide(APS),in vaccination control(VC)、virus control group and BC group with physiological saline,once a day for three successive days.On days 28 after the first vaccination,the chickens were attacked with New castle disease virus(NDV).The symptom and the death time were recorded,and the morbidity,mortality and protection rate were calculated.The results showed that the morbidities and mortalities of GLP at high and middle dose were significantly lower than that of the virus control group(P<0.05),slightly,but not significantly lower than that of the APS control group and the vaccination group.The results indicated that GLP could increase the immune protective effects of Newcastle disease vaccine for chickens,and increase the antiviral action of the body.更多还原