【作者】 冯伟；

【导师】 李健；

【作者基本信息】 上海海洋大学 ， 临床兽医学， 2011， 硕士

【摘要】 维生素C和维生素E是两种在维持水产动物健康养殖中应用较广的免疫增强剂,裂壶藻是一种富含不饱和脂肪酸的海洋真菌,本文通过结合免疫应答信号通路的相关基因的研究,探索两种维生素在机体中的免疫途径,初步研究裂壶藻与维生素E的免疫互作效应,为其免疫机制研究提供新的思路。本文以中国对虾为研究对象,在不同水平上研究了维生素C、维生素E和裂壶藻对中国对虾非特异免疫因子及TLR、NF-κB表达水平的影响。研究分为两部分:第一部分是以中国对虾为实验动物,对其投喂分别添加不同Vc含量(0%、0.5%、1%、2%)的饲料,在试验的第1、3、6、9、12和15天进行取样,以存活率、诱导型一氧化氮合成酶(iNOS)、过氧化氢酶(CAT)、总超氧化物歧化酶(T-SOD)、溶菌酶(LZM)为免疫指标,探讨了不同浓度的Vc对中国对虾存活率及非特异性免疫效应的影响;并应用实时定量PCR方法研究了不同浓度的Vc对中国对虾TLR、NF-κB基因表达的影响。结果表明,(1)与对照组相比,Vc添加组增强了中国对虾的存活率,Vc1%添加组存活率达90.28%,显著高于其他组(P<0.05)。Vc的不同处理组在一定程度上提高了中国对虾iNOS、CAT、及LZM活性且1%含量Vc组对酶活性的增强影响较大。Vc对中国对虾T-SOD活性的提高具有一定的作用,2%浓度组对肝胰腺、鳃、肌肉T-SOD活性影响较大,1%浓度组对血清中T-SOD活性影响较大(2)实时定量PCR结果表明,Vc可以上调TLR在血液和鳃中TLR的表达水平。血液中1%浓度组对TLR的表达水平的上调作用最大,其次为0.5%浓度组。鳃中Ve对TLR的上调作用具有一定的浓度效应,即表达水平2%浓度组>1%浓度组>0.5%浓度组。在肝胰腺中Vc对0.5%、1%浓度组TLR的表达水平起到抑制作用,而对2%浓度组TLR则是先抑制后上调,在第十二天表达量达到最大值,为对照组的6.87倍。肌肉中Vc对2%浓度组TLR mRNA表达具有下调作用,对0.5%、1%浓度组TLR的表达呈现出先抑制后促进的状况。TLR、NF-κB在组织中的表达趋势并不一致,0.5%、2%浓度组在四种组织中均表现出下调作用。1%浓度组在血液、肌肉中对NF-κB的调节作用相同,与各自对照组相比均可以上调NF-κB的表达水平。而在鳃、肝胰腺中1%浓度Vc所起到的作用与血液和肌肉相反。第二部分是以中国对虾为实验动物,在基础饲料中添加不同含量的Ve和10裂壶藻,配制成五种试验饲料,即B组(10裂壶藻)、C组(200mg/kgVe)、D组(400mg/kgVe)、E组(600mg/kgVe)、F组(400mg/kgVe+10裂壶藻),以基础饲料为A组(空白对照饲料)。进行为期1个月的饲养。在试验第1、15、30天进行体重、体长测量,检测Ve对中国对虾生长指标的影响。记录每日对虾死亡数,统计存活率。并以组织中的诱导型一氧化氮合成酶(iNOS)、丙二醛(MDA)、总超氧化物歧化酶(T-SOD)、溶菌酶(LZM)、谷胱甘肽酶(GSH)为免疫指标,探讨了不同浓度的Ve对中国对虾存活率及非特异性免疫效应的影响;应用实时定量PCR方法研究了不同浓度的Ve对中国对虾TLR、NF-κB基因表达的影响。结果表明,(1)Ve可以促进中国对虾体重、体长的增长。D组生长指标增长高于C、E组,但显著低于添加了裂壶藻的F组。存活率随着Ve添加浓度的增加而增大,E、F组显著高于其他各组。说明Ve对中国对虾的生长及存活率的提高有着显著的作用。添加裂壶藻也对生长和存活率的提高起到促进作用。通过对中国对虾非特异性酶活的检测,结果表明,与空白对照组相比,Ve抑制了MDA活性、肌肉中iNOS活性。对于其他非特异酶均表现出不同程度的促进作用。裂壶藻添加组B组与A组相比较非特异性酶活性无显著性差异,但与F组表现出显著性差异(P<0.05),即F组酶活性显著高于B组。(2)应用实时定量PCR方法研究了不同浓度的Ve对中国对虾血液、肌肉及肝胰腺中TLR、NF-κB基因表达的影响。结果表明,Ve可以下调血液、肝胰腺和肌肉中的TLR表达水平,并具有一定的浓度效应;中浓度Ve对三个组织NF-κB基因的表达均起到抑制作用。添加10裂壶藻后, A、B、D、F组TLR表达水平在肝胰腺和肌肉中的趋势相同,表达水平为A>B>D>F;NF-κB基因表达在血液和肝胰腺中的趋势相同,表达水平为B>A>D>F,说明饲料中添加一定量的裂壶藻可以降低机体内TLR、NF-κB基因的表达水平,Ve和裂壶藻联合对免疫基因的调控能力更加显著。更多还原

【Abstract】 Vitamin C and vitamin E are two immunopotentiators applied widely in the aquatic animal health cultivation. Schizochytrium is a Marine fungi , rich in unsaturated fatty acid .In order to providing new thinking for immune mechanism of vitamin, the genes related to immune response signaling pathway and the immune path the two kinds of vitamin were researched in this paper. The Chinese shrimp was used for experimental object in this experiment. The effects of the nonspecific immunity factors and TLR and NF-κB mRNA expression of Chinese shrimp at different concentrations of vitamin C and vitamin E were researched in this paper. The research was divided into two parts:The Chinese shrimp was used for experimental animal in this experiment in the first part. We fed different leVels (0%,0.5%,1%,2%) of Vc to study the influence on the Chinese shrimp.After 1, 3, 6, 9, 12 and 15 days, We sampled and detected survival rate and activity of inducible nitric oxide synthase (iNOS), catalase (CAT), total superoxide dismutase (T-SOD) and lysozyme (LZM), taking which as immune indexs, the nonspecific immunity effects at different concentrations were inVestigated in this paper. We also used real-time fluorescent quantitatiVe PCR (RT-PCR) to examine the expression TLRs and NF-κB genes in the Chinese shrimp. Results showed that: (1) Comparing to control group, all Vc groups could raise the survival rate. The 1% group showed the highest survival rate among groups,with survival rate at 90.28%, significantly higher (P<0.05) than that of other groups. All Vc groups could raise the activity of iNOS,CAT and LZM at some degree and 1% group showed the best immune activating effects among groups. All Vc groups also could raise the activity of T-SOD, 1% group showed the best immune activating effects on T-SOD in serum ,the SOD activity in the gills, muscle and hepatopancreas of the Vc group of high concentration, especially the hepatopancreas, showed higher than that of other groups.In a word, the survival rate and the activity of nonspecific immunity enzyme were the best in the 1% Vc group. (2) RT-PCR showed that, Vc had up-regulatory activity on mRNA expression of TLR and NF-κB. 1% Vc group had the most significant up-regulatory activity on mRNA expression of TLR in the serum. And 0.5% Vc group was in the next. The up-regulatory activity of Vc on mRNA expression of TLR in the gill was related to concentration of Vc. The leVels of mRNA expression of TLR were 2% group>1% group>0.5% group. The mRNA expression of TLR was inhibited in the 1% group and 0.5% group, though, which was inhibited first and then promoted and reached the maximum after 12 days which was 6.87 times of control group. 2% Vc group had down-regulatory activity on mRNA expression of TLR in the muscle. The mRNA expression of TLR of 1% group and 0.5% group showed inhibition first and then promotion. The leVels of TLR and NF-κB mRNA expression in the tissue were different. 0.5% and 2% Vc group both had down-regulatory activity on mRNA expression of TLR and NF-κB in the four tissue. NF-κB mRNA expression in the serum and muscle of 1% group is higher than that in other groups. HoweVer, the actions in the gill and hepatopancreas of the 1% group were on the contrary.The Chinese shrimp was also used for experimental animal in this experiment in the next part. Different concentrations Ve and Schizochytrium were added into basic feedstuff. There were 5 groups, including B group(10% Schizochytrium), C group(200 mg/kg Ve), D group(400 mg/kg Ve), E group(600 mg/kg Ve), and F group(400 mg/kg Ve + 10% Schizochytrium). The A group (basic feedstuff) was control group. The experiment lasted 1 month. After 1, 15 and 30 days, we sampled and detected body weight and length. The death rates and survival rates were checked eVeryday. The activity of inducible nitric oxide synthase (iNOS), total superoxide dismutase (T-SOD), lysozyme (LZM), malondialdehyde (MDA) and glutathione (GSH) were taked as immune indexs to inVestigate the nonspecific immunity effects at different concentrations of Ve. The real-time fluorescent quantitatiVe PCR (RT-PCR) was used to examine the expression TLRs and NF-κB genes in the Chinese shrimp. The results showed that: (1)Comparing to control group, all Ve groups could promote the increase of body weight and length, which of D group was higher than C group and E group while obviously lower than F group. The higher concentration of Ve, the higher survival rate. The survival rates of E group and F group were significantly higher than that of other groups, which indicated that Ve is beneficial to growth and survival rate of the Chinese shrimp. There was no significantly different between A group and B group about the nonspecific immunity enzyme. HoweVer, the nonspecific immunity enzyme of F group was significantly higher than that of B group. (2) The mRNA expression of TLR was inhibited by Ve in the muscle and hepatopancreas. Comparing to control group, 200 mg/kg Ve group could reduce the leVel of TLR on serum and other groups had up- regulatory activity , 200 mg/kg Ve group had down-regulatory activity on mRNA expression of NF-κB in the three kinds of tissue. For A, B, D and F group, the tendency of TLR mRNA expression in the muscle was the same as that of hepatopancreas, whose order of expression leVel was A>B>D>F. And the tendency of NF-κB mRNA expression in the serum was the same as that of hepatopancreas, whose order of expression leVel was B>A>D>F.It indicated that increasing some Schizochytrium in the feedstuff would reduce leVels of NF-κB and TLR mRNA expression. Combining Ve and Schizochytrium could more effectiVely improVe the regulation of immune genes.更多还原