【作者】 王艳萍；

【导师】 祁克宗；

【作者基本信息】 安徽农业大学 ， 基础兽医学， 2006， 硕士

【摘要】 本研究选取20日龄三黄肉仔鸡为研究对象,随机分为四组,实验组A(灌服LPS 100mg/Kg)、B(灌服LPS 200mg/Kg)、C(灌服LPS 300mg/Kg)和对照组D(灌服生理盐水)。应用病理学、生物化学及分子生物学等方法,检测不同剂量的LPS在不同时间对肉仔鸡肝、肠组织以及内毒素识别因子肝脏钟样受体4(TLR4) mRNA表达的影响,为进一步揭示内毒素在幼禽肠道革兰氏阴性菌感染和细菌易位所引起消化器官损伤的机制提供依据。1.内毒素对肉仔鸡肠、肝形态变化的影响肠道主要病理变化为肠壁变薄、肠绒毛肿胀,炎性细胞浸润等;对肝脏主要病理变化为肝细胞变性、淤血以及炎性细胞浸润等。本实验结果表明,低剂量内毒素对幼禽影响较小,随着剂量的增加,其损伤程度加深。2.内毒素对肉仔鸡血液生化指标的影响为观察内毒素对肝脏功能的影响,检测肉仔鸡血液生化指标:天冬氨酸转氨酶(AST)和丙氨酸转氨酶(ALT)活性、直接胆红素(DBIL)、间接胆红素(IBIL)。实验表明不同剂量LPS对实验鸡血液的生化指标有不同程度的影响,结果如下:实验各组血清AST变化:总的趋势是先升高后回落,其中A组4h、12h和48h与对照组相比差异极显著(P<0.01),而12h后逐渐回落,B组4h、48h和C组4h、12h、48h与对照组比较差异极显著,(P<0.01),B组12h、24h与对照组比较差异显著。(P<0.05)实验各组血清ALT活性变化:总的趋势是先升高后降低,其中A组4 h和12h与对照组相比差异显著(P<0.05),而12h后逐渐恢复正常,B、C两组各时间点与对照组相比差异都是极显著(P<0.01)实验各组血清DBIL含量变化:A、B组呈先升高后降低的趋势,C组达到峰值后持续,A组4h和12h以及B、C两组各时间点与对照组比较差异极显著(P<0.01)。实验各组血清IBIL变化,A、B组呈现先升高后降低的趋势,其中A组48h时能恢复至攻毒前的高水平,而B组达峰值后持续高水平,C组一直处于较高水平。其中A、C组与对照组比较各时间点差异均极显著(P<0.01),B组与对照组比较仅在24 h差异显著(P<0.05)。3.内毒素对肉仔鸡抗氧化酶的影响用黄嘌呤氧化酶法、硫代巴比妥比色法分别检测肉仔鸡血浆超氧化物歧化酶(SOD)活性、丙二醛(MDA)水平。实验表明,给肉仔鸡喂服不同剂量的内毒素后24 h内可明显降低小鸡血液中SOD的活性,鸡体的抗氧化功能失常。结果如下:实验各组血浆SOD活性:实验鸡喂服内毒素后,各组均表现为先降低后升高的趋势,A组SOD活性于12h降到最低值,以后逐渐回升到接近正常值,B组于各剂量组血浆中SOD活性表现为先降低后升高的变化规律,灌服内毒素后24h显著降低(P<0.05),但于24h后则呈升高趋势。实验各组血浆MDA:实验鸡灌服LPS后,各剂量组血浆中MDA含量表现为先升高后降低的趋势,不同剂量组之间同一时间点变化幅度随着剂量的增加而升高,除C组在48h升至最高值外,A组和B组都是在24h升至最高值。4 .内毒素对肉仔鸡肝脏TLR4 mRNA表达的影响为观察内毒素对实验鸡肝脏TLR4 mRNA表达的影响,应用RT-PCR技术,动态检测不同时间点实验鸡肝脏TLR4 mRNA表达的变化: A组稍高于D组,B、C组于12h降至最低值,但相对于A、B组来说,C组数值较低。实验表明,TLR4 mRNA表达呈时间依从性,LPS信号转导分子TLR4在介导LPS肝损伤中起重要用。更多还原

【Abstract】 Ninety-six chickens twenty-day-old, SanHuang, were divided into four experiment groups randomly, In order to search for the injury mechanism in poultry, Group One of chicken were orally given physiological brine as control (D), the other three groups of chicken were orally given lipopolysaccharide, group two 100mg/kg (A),group three 200mg/kg (B), group four 300mg/kg (C) respectively. Methods of pathology, biochemistry and molecule biology were used to examine the injury in liver, small intestine and TLR4 mRNA expression of the liver in different phase in chicken. Hope to provide adequate grounds for injury evoked by LPS on the infection of G- bacteria and bacterial translocation in intestine of poultry.1. Effect of LPS on morphologic change of liver and intestine in chicken Injury of liver: Liver cell degeneration, hyperemia and macrophase illilization,etc. Injury of intestine: small intestine wall was thinner than group D, swelling of small intestine villus, leukocytosis of small intestine,etc.Experiment indicated lower dose of LPS had light injury on chicken,and higer dose of LPS had heavy injury on chicken.2. Influence of the blood biochemical paramerer of chicken treated with LPS In order to observe the hepatic functions of experiment chickens treated with LPS, Activies of AST, ALT, levels of DBIL, IBIL and ratio of ALB/GLO were detected. This study showed that different doses of LPS had different degrees damnification to experiment chickens.Results as following:Changes of AST in serum,it increased early time, decreased latterly. The activies of 4 h,12 h, 48 h in group A , 4 h, 48 h in group B and 4 h,12 h, and 48 h were significant compared with group D.(P<0.01)It was significant that in 12 h and 24 h in group B comared with group D.(P<0.01)Changes of ALT in serum, it increased early time, decreased latterly, the difference between 4 h and 12 h in group A was significant (P<0.05), and returned 12 h later, the differences between group B, C and D was significant (P<0.01).Changes of DBIL in serum,the tedency in group A, B is increased in early time and decreased lately, and group C went on. The difference between group A of 4 h,12 h and group D was significant (P<0.01), the diffeces beteen group B, C and group D both were significant. (P<0.01)Changes of IBIL in serum,the tendency was increased in early time and decreased laterly in group A and B,group A could return normal among them, but group B went on at high level after reaching top. The difference beteen group A, C and D was significant (P<0.01), the difference between group B and group D was significant only at 24 h. (P<0.05)3. Influence on anti-oxygenation enzyme for the chicken treated with LPSThere was no difference in control group on SOD, after given LPS, the tedency decreased in early time and increased laterly in experiment group A、B、C. In group A the activities of SOD decreased to the lowest at 12 h,the increased near to the normal level. In experiment group B,the lowest level was reached at 24 h,, then returned. It showed that after given different dose of LPS to the chickens, LPS could reduce the activities in 24 h, large amount of LPS can damnify chicken.LPS could increase the content of MDA in blood plasama. The tendency was increased at early time and returned lately after given LPS, and it increased with the dose of LPS at a certern time.Top level was reached at 24 h in group A and B, but group C was at 48 h.4. TLR4 mRNA Expression for liver treated with LPSIn order to observe the inlluence of LPS on TLR4 mRNA Expression for liver, RT-PCR technology was used to detect the TLR4 mRNA expression at indicated timepoints. Group A was higher than group D a little, group B, C decreased to the lowest at 12 h, but group C had the lowest number compared with group A, B. This study showed that TLR4 mRNA Expression changed with time and TLR4 had important role in LPS mideated liver injury.Study showed that different damnification can be made in chicken liver by LPS, and it got deeper with the dose of LPS.更多还原