【作者】 罗晓容；

【导师】 吴德；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2009， 硕士

【摘要】 本试验旨在研究不同维生素A(Vitamin,VA)水平对感染伪狂犬病毒(PseudorabiesVirus,PRV)雌鼠妊娠第9天免疫功能及繁殖性能的影响。VA缺乏模型建立后,选择体重为36.65±7.35(g)的160只雌鼠按照不完全配对试验设计分到8个处理组:其中5个VA水平(0 IU/kg、4000 IU/kg、10000 IU/kg、25000 IU/kg、50000 IU/kg)为PRV攻毒组,3个VA水平(0 IU/kg、4000 IU/kg、10000 IU/kg)为注射PBS的对照组。雌鼠于妊娠0天腹腔注射PRV或PBS。妊娠第9天屠宰收集孕鼠血清、脑、肝脏、脾脏、子宫和胚胎。利用HPLC测定肝脏中视黄醇浓度:普通PCR法检测脑组织PRV感染情况;计算脾脏指数;利用透射免疫比浊法测定血清免疫球蛋白(IgG、IgA、IgM)浓度;利用ELISA法测定血清细胞因子(IL-1β、IFN-γ、IL-4、IL-10)浓度;利用RT-PCR方法检测目的基因Toll样受体3(Toll-like receptor 3,TLR3)、7、9 mRNA在子宫和胚胎中的表达量。结果表明:1、肝脏中视黄醇浓度随VA水平的增加而增加。攻毒后,50000 IU/kg组孕鼠肝脏中视黄醇浓度显著高于其它VA水平组(P＜0.05)。攻毒组肝脏中视黄醇浓度低于PBS组,且4000 IU/kg攻毒组视黄醇浓度显著低于相同VA水平下PBS组(P＜0.05)。2、攻毒后,VA缺乏组脾脏指数显著低于其它VA水平组(P＜0.05)。注射PBS条件下,0 IU/Kg和4000 IU/Kg组脾脏指数显著低于10000 IU/Kg组(P＜0.05)。4000IU/Kg攻毒组脾脏指数显著高于相同VA水平下PBS组(P＜0.05)。3、攻毒后,50000 IU/Kg组血清IgA和IgM水平显著高于0 IU/Kg组(P＜0.05)。除VA缺乏组外,IgG水平随VA水平的增加而增加,4000 IU/Kg组显著低于10000IU/Kg组、25000 IU/Kg组和50000 IU/Kg组(P＜0.05)。注射PBS条件下,IgA、IgM和IgG水平随VA水平的增加而增加。攻毒组IgA和IgM水平低于PBS组,IgG水平高于PBS组。4、补充VA有降低血清IL-1β、IFN-γ水平而增加IL-4、IL-10水平的趋势。攻毒后,25000 IU/Kg组血清IL-1β水平显著低于其它VA水平组(P＜0.05)。10000 IU/Kg组、25000 IU/Kg组和50000 IU/Kg组IFN-γ水平显著低于0 IU/Kg组和4000 IU/Kg组(P＜0.05)。25000 IU/Kg组IL-4水平显著高于其它VA水平组(P＜0.05)。25000 IU/Kg组IL-10水平显著高于0 IU/Kg组(P＜0.05)、4000 IU/Kg组(P＜0.05)和10000 IU/Kg组(P＜0.05);25000 IU/Kg组和50000 IU/Kg组间IL-10水平无显著差异(P＞0.05)。注射PBS条件下,10000 IU/Kg组IL-1β、IFN-γ水平显著低于0IU/Kg组(P＜0.05)。血清IL-4和IL-10水平随VA水平的增加而增加,其中10000 IU/Kg组血清IL-4水平显著高于0IU/Kg组(P＜0.05)。攻毒组血清1L-1β、IFN-γ水平高于PBS组,血清IL-4和IL-10水平低于PBS组。5、补充VA降低妊娠9天子宫和胚胎TLR3、TLR7、TLR9相对表达量。攻毒后,25000 IU/Kg和50000 IU/Kg组子宫及胚胎TLR3、TLR7、TLR9相对表达量显著低于0 IU/Kg(P＜0.05)和4000 IU/Kg(P＜0.05)组。注射PBS条件下,10000 IU/Kg组子宫和胚胎TLR3、TLR9及胚胎TLR7相对表达量显著低于0 IU/Kg组(P＜0.05)。攻毒组子宫和胚胎TLR3、TLR7、TLR9相对表达量高于PBS组。6、攻毒后,VA缺乏增加孕鼠死亡率和流产率,补充VA有降低死亡率和流产率趋势(P＞0.05)。7、攻毒后,25000 IU/Kg组妊娠9天活胚数和分娩活仔数显著高于0 IU/kg组(P＜0.05)。活胚数随VA水平增加呈二次曲线变化,VA添加量为34265 IU/Kg时(正常需要量的8.5倍)达最大值。注射PBS条件下,妊娠9天活胚数和分娩活仔数随VA水平的增加而增加。攻毒组活胚数和分娩活仔数低于PBS组,且4000 IU/Kg攻毒组活胚数显著低于相同VA水平下PBS组(P＜0.05)。结论:1、VA临界缺乏状态下,感染PRV导致死亡率和流产率增加,活胚数和分娩活仔数降低;补充VA可降低孕鼠死亡率、流产率,提高妊娠9天活胚数和分娩活仔数;妊娠第9天活胚胎数达最大值时,孕鼠对VA的需要量达正常需要量的8.5倍。2、VA缺乏的孕鼠感染PRV后上调TLR3、TLR7、TLR9 mRNA相对表达量,加剧Th1型促炎症细胞因子的表达,影响Th1/Th2免疫平衡状况,不利于动物繁殖性能的发挥;补充VA可降低TLR3、TLR7、TLR9 mRNA相对表达量,提高Th2型细胞因子的表达,改善Th1/Th2免疫平衡,从而对胚胎发育提供免疫保护效应。3、补充VA可提高感染PRV孕鼠血清免疫球蛋白水平,提高孕鼠体液免疫功能,并改善胚胎发育。更多还原

【Abstract】 This study was conducted to investigate effects of vitamin A on immunity and reproductive performance on 9^th pregnancy of mice infected Pseudorabies Virus.The mice model with vitamin A deficiency was established,160 mice weighted 36.65±7.35（g） were assigned to 8 treatments according to an incomplete paired design.Mice in the three control groups were injected with PBS and supplied with vitamin A at 0,4000 and 10000 IU/kg, respectively;mice in the five experimental groups were infected Pseudorabies Virus and supplied with Vitamin A at 0,4000,10000,25000 and 50000 IU/kg,respectively.Female mice were injected Pseudorabies Virus or PBS on 0^th pregnancy and mice were killed on 9^th pregnancy to collect serum、brain、liver、spleen、uterus and embryos.Hepatic retinol concentration was determinted by HPLC,Ordinary PCR was used to determine PRV infection in brain,calculation spleen index,Immunity Transmission Turbidimetry was applied to determine maternal immunoglobulins（IgG、IgA、IgM） concentration in serum,ELISA was applied to determin cytokine（IL-1β、IFN-γ、IL-4、IL-10） concentration in serum,and real time-quantitative RT-PCR was applied to determine the expression of genes（TLR3、TLR7、TLR9） in uterus and embryos.The results were as follows:1、Hepatic retinol concentration increased with vitamin A level and was lower in the challenged than in the PBS group.Among challenged groups,hepatic retinol concentration in the 50000 IU/kg group was significantly higher than other vitamin A groups（P＜0.05）, and between 4000 IU/kg groups,it was significantly lower in the challenged than in the PBS group（P＜0.05）.2、Among challenged groups,spleen index was significantly lower in the 0 IU/kg than in other groups（P＜0.05）,among PBS groups,it was significantly lower in the 0 and 4000 IU/kg group than in the 10000 IU/kg group（P＜0.05）.and between 4000 IU/kg groups,it was significantly higher in the challenged than in the PBS group（P＜0.05）. 3、Among challenged groups,IgA and IgM concentration in the 50000 IU/Kg group was significantly higher than in the 0IU/Kg group（P＜0.05）.IgG concentration increased with vitamin A level except 0 IU/Kg group,and it was significantly lower in the 0 IU/Kg group than in the 10000 IU/kg,25000 IU/Kg and 50000 IU/Kg group（P＜0.05）.Among PBS groups,IgA、IgM、IgG concentration increased with vitamin A level increased.Compared with PBS groups,IgG concentration increased and IgA、IgM concentration decreased in challenged groups at the same vitamin A level.4、With vitamin A level increased,IL-1β、IFN-γconcentration tended to decrease and IL-4、IL-10 concectration tended to increase.Among challenged groups,IL-1βconcentration in the 25000 IU/Kg group was significantly lower than in other vitamin A groups（P＜0.05）. IFN-γconcentration in the 10000 IU/Kg group、25000 IU/Kg group and 50000 IU/Kg group were significantly lower than in the 0 IU/Kg and 4000 IU/Kg group（P＜0.05）.IL-4 concentration in the 25000 IU/Kg group was significantly higher than in other vitamin A groups（P＜0.05）.IL-10 concentration in the 25000 IU/Kg group was significantly higher than in the 0 IU/Kg、4000 IU/Kg and 10000 IU/Kg group（P＜0.05）,and there were no significant differences between 25000IU/Kg group and 50000 IU/Kg group（P＞0.05）. Among PBS groups,IL-1β、IFN-γconcentration in the 10000 IU/Kg group was significantly lower than in the 0 IU/Kg group（P＜0.05）.IL-4 and IL-10 concentration increased with vitamin A level increased,and IL-4 concentration in the 10000 IU/Kg group was significantly higher than in the 0 IU/Kg group（P＜0.05）.Compared with PBS groups, IL-1β、IFN-γconcentration increased and IL-4、IL-10 concentration decreased in challenged groups at the same vitamin A level.5、With vitamin A level increased,expression of TLR3/TLR7/TLR9 decreased.Among challenged groups,they were significantly lower in the 25000 IU/Kg and 50000 IU/Kg group than in the 0 IU/Kg and 4000 IU/Kg group（P＜0.05）.Among PBS groups,expression of TLR3、TLR9 in uterus and embryos and TLR7 in embryos in the 10000 IU/Kg group were significantly lower than in the 0 IU/Kg group（P＜0.05）.Expression of TLR3、TLR7、TLR9 was higher in challenged group than in the PBS group at the same vitamin A level.6、Morality and abortion rate increased postchallenged,and tended to decrease with vitamin A level increased（P＞0.05）. 7、Among challenged groups,live embryos and live litter size in the 25000 IU/Kg group were significantly higher than in the 0 IU/Kg group（P＜0.05）.Regression analysis revealed that there was a quadratic increase in live embryos on 9^th pregnancy,with the highest values observed at 34265 IU/kg（8.5 times that of mice with normal live embryos）.Among PBS group,live embryos on 9^th pregnancy and live litter size increased with vitamin A level increased.Live embryos and live litter size decaresed when infected PRV,and between 4000 IU/kg groups,it was significantly lower in the challenged than in the PBS group（P＜0.05）.Conclusion:1、When vitamin A level was in marginal deficiency,Pseudorabies Virus infection resulted in increased abortion rate and mortality and decreased live embryos and live litter size;However,abortion rate and mortality decreased and live embryos and live litter size increased when supplied vitamin A,and vitamin A requirement of female mice with highest live embryos on 9^th pregnancy was 8.5 times that of mice with normal live embryos.2、When vitamin A level was in marginal deficiency,Pseudorabies Virus infection resulted in increased expression of TLR3、TLR7、TLR9,then aggvarated expression of Th1 inflammatory factors,then affected Th1/Th2 immune balance,which was harmful to reproductive performance;However,expression of TLR3、TLR7、TLR9 decreased and level of Th2 antiinflammatory factor increased when supplied vitamin A,then Th1/Th2 immune balance improved which provided protective effect to embryonic development.3、Spplied vitamin A to pregnant mice infected PRV can increase serum immunoglobulin concentration,increase humoral immunity and improve embryos development.更多还原