【作者】 邱时秀；

【导师】 吴德； 方正峰；

【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】 母体的免疫特别是Th1/Th2平衡对妊娠成功与否起决定作用,母体感染疾病后Th1型促炎症细胞因子增加,破坏Th1/Th2平衡不利于妊娠,而色氨酸的代谢产物可以调控Th1和Th2细胞因子的比例,从而影响繁殖。因此,本研究以妊娠雌鼠为模型,以动物繁殖性能(死亡率、流产率、活胚数、分娩活仔数)、第9天脾脏指数、免疫球蛋白(IgG、IgA、IgM)、细胞免疫(细胞因子IL-1β、IL-10、IFN-γ)、孕酮、子宫和胚胎IDO、Toll样受体(Toll-like receptor,TLR)及第9天和分娩时伪狂犬病特异性抗体为评价指标,旨在探讨不同色氨酸水平对雌鼠繁殖性能的影响及其可能的机制。试验选用体重为176只(34±5.5g)的雌鼠。按体重随机分成8组,每个处理7个重复,每个重复3-4只。其中4个Trp水平(0.2%、0.35%、0.5%、0.75%)为伪狂犬病毒(pseudorabies virus,PRV)攻毒组,对应的4个Trp水平(0.2%、0.35%、0.5%、0.75%)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136只,余下自然分娩。试验全期自由采食,自由饮水。1.提高色氨酸水平改善孕鼠繁殖性能。0.2%攻毒组死亡率与流产率显著高于0.35%和0.5%攻毒组。0.35%、0.5%、0.75%攻毒组第九天活胚数显著高于0.2%攻毒组(P＜0.05),且0.5%攻毒组活胚数最高,而PBS组活胚数差异不显著(P＞0.05)。用活胚数进行回归分析,在攻毒情况下色氨酸水平为0.561%时活胚数最高。补充色氨酸提高血清孕酮水平。0.35%、0.5%攻毒组血清中孕酮水平显著高于0.2%组(P＜0.05)。2.色氨酸水平影响孕鼠妊娠第9天脾脏指数及免疫相关因子的表达。提高色氨酸水平可以改善脾脏发育。0.2%和0.75%攻毒组脾脏指数显著低于0.5%水平组(P＜0.05)。0.5%攻毒组脾脏指数显著高于同一色氨酸水平下PBS组。补充色氨酸可显著增加血清中9天和分娩时伪狂犬病毒特异性抗体阻断率、IgG、IgM的浓度(P＜0.05),而对IgA影响差异不显著。无论是妊娠第九天还是分娩时0.5%攻毒组抗体阻断率显著高于0.2%组(P＜0.05)。攻毒组随色氨酸浓度的增加升高血清IgG浓度。0.35%、0.5%攻毒组血清IgG的浓度显著高于0.2%攻毒组,而0.5%攻毒组IgG浓度又高于0.35%组。0.5%攻毒组IgM的浓度显著高于其余各组。补充色氨酸可降低胚胎组织中IL-1β、IFN-γ水平而增加IL-10水平。0.5%攻毒组胚胎IL-1β水平显著低于0.2%、0.75%色氨酸攻毒组(P＜0.05)。0.2%攻毒组胚胎IFN-γ水平显著高于其余各水平攻毒组(P＜0.05)。攻毒显著增加胚胎IFN-γ水平(P＜0.05)。0.5%、0.35%攻毒组胚胎IL-10水平显著高于其余两组(P＜0.05)。攻毒显著减低胚胎IL-10水平(P＜0.05)。补充色氨酸降低妊娠9天子宫和胚胎TLR3、TLR9相对表达量,升高子宫和胚胎IDO的表达量。0.2%攻毒组子宫及胚胎IDO相对表达量显著低于其余各水平攻毒组。0.35%和0.5%攻毒组子宫及胚胎TLR3、TLR9相对表达量显著低于0.2%攻毒组(P＜0.05)。攻毒组子宫和胚胎TLR3、TLR9相对表达量高于PBS组。结论:1、攻毒提高孕鼠死亡率和流产率,降低胚胎存活率和活产仔数,添加色氨酸可降低攻毒孕鼠的死亡率和流产率,提高胚胎存活率和活产仔数。其机制在于添加色氨酸调节病毒感染孕鼠子宫内环境Th1/Th2的比例,并下调TLR基因的表达。2、攻毒提高孕鼠的色氨酸需要量,保证感染PRV孕鼠第9d活胚胎数最高时,色氨酸的需要量为正常孕鼠的1.6倍。更多还原

【Abstract】 It has been established that the maternal immune response,particularly the balance of T-helper type-1（Th1）/Th2 cytokine,plays a key role in determinating the success or failure of pregnancy.Virus infection resulted in increased synthesis of Th1-type cytokines and induced abortion or birth of dead.It has been shown that catabolites of tryptophan,induce apoptosis in Th1 cells,but not in Th2 cells.This study therefore examined the effect of dietary tryptophane level[low（0.2%, 0.8×maintenance）,medium（0.35%,1.4×maintenance）,high（0.5%and 0.75%, 2×maintenance and 3×maintenance,respectively）]on the immunity and reproduction of PRV-challenged pregnant mice,with mice fed the same diet but injected equal volume of phosphate buffered saline（PBS） at the same time points of pregnancy serving as a control.Blood samples,uterus and embryos on the 9th d of pregnancy were collected for determination of mRNA abundance and concentrations of cytokine,progesterone,immunoglobulins and PRV-specific antibody.The results were as follows:Tryptophane supplements improved pregnancy success.The mortality and abortion rate in 0.2%PRV group was significantly higher than in 0.35%PRV and 0.5%PRV group（P＜0.05）.In PRV groups,dietary inclusion with 0.35%,0.50%and 0.75%tryptophane all resulted in significantly（P＜0.05） increased live embryos compared with the 0.2%group,with highest live embryos observed in the 0.50% group,whereas the number of live embryos did not differ（P＞0.05） among the PBS groups.According to the regression equation,the maximal number of live embryos could be obtained by dietary tryptophane inclusion of 0.561%diet.The maximal live litter size in PRV groups was observed in the 0.5%group.Higher（P＜0.05） was progesterone concentrations observed in 0.35%and 0.5%groups than in 0.2% groups.Tryptophane supplements affected immune on 9^th pregnancyIn PRV groups, spleen index was significantly lower in the 0.2%and 0.75%groups than in the 0.5% groups（P＜0.05）.Spleen index was significantly（P＜0.05） higher in 0.5%PRV than in 0.5%PBS group.High dosage tryptophane resulted in promoted PRV-specific antibody synthesis and increased immunoglobulin（Ig） G,IgM,Blocking rate of specific antibody measured either on the 9th day of pregnancy or on delivery day were significantly（P＜0.05） higher in PRV groups than in PBS groups.Higher （P＜0.05） blocking rate of PRV-specific antibody was observed in 0.5%groups than in 0.2%groups either on the 9th day of pregnancy or on delivery day.Higher （P＜0.05） IgG concentration was observed in 0.35%and 0.5%PRV groups compared with that in 0.2%PRV groups.IgG concentrations of 0.5%PRV groups was also higher（P＜0.05） than that of 0.35%PRV groups.In PRV groups,preganant mice fed 0.5%tryptophane had the highest（P＜0.05） serum IgM concentration.Both IL-1βand IFN-γconcentration were significantly（P＜0.05） decreased with the increasing of dietary tryptophane level.IL-10 concentration were significantly（P＜0.05） increased with the increasing of dietary tryptophane level from 0.2%to 0.5%PRV groups.Each treatment group,IFN-γwas significantly（P＜0.05） lower,in PBS than in PRV groups.IDO expression of PRV-infected groups were significantly（P＜0.05） increased with the increasing of dietary tryptophane level either in the embryo or in the uterus. In the PBS groups,IDO expression of 0.2%groups was lower than other groups in embryo（P＜0.05）.Compared with the 0.2%PRV group,the 0.35%and 0.5%PRV group had significantly（P＜0.05） lower TLR3 and TLR9 expression either in the embryo or in the uterus,but increase was observed with the increasing of dietary tryptophane from 0.5%to 0.75%.PRV infection also caused the effect on TLR expression.Receiving the same dosage of tryptophane,PRV-infected mice had higher uterine expression of TLR3 and TLR9 than PBS-injected mice.In conclusion,PRV challenge resulted in increased tryptophan requirement by pregnant mice.The 0.561%tryptophan level had better embryonic survival compared with other groups.Tryptophan levels affected embryo survival of PRV-infected pregnant mice by down-regulating TLR expression and proinflammatory cytokine synthesis,up-regulating the PRV-specific antibodies, anti-inflammatory cytokine and progesterone synthesis and concentrations of immunoglobulins that were beneficial to resist virus invasion and embryonic development.更多还原