【作者】 张华；

【导师】 黄克和； 薛家宾；

【作者基本信息】 南京农业大学 ， 临床兽医， 2004， 硕士

【摘要】 大量研究报道，硒能影响免疫系统各个部分的功能，家畜、家禽及人缺硒其非特异性免疫、体液免疫及细胞免疫功能均会不同程度地受到抑制，若给正常和缺硒畜禽补充适量的硒则能增强免疫应答反应。但迄今为止，硒对兔免疫功能的影响未见系统研究报道，而用不同硒源同时研究硒对动物细胞免疫和体液免疫功能的影响的材料较少见到。本试验选用亚硒酸钠、硒化卡拉胶和硒代蛋氨酸3种不同硒源，从体外到体内研究其对兔细胞免疫和体液免疫功能的影响，同时比较了亚硒酸钠、左旋咪唑与黄芪多糖对兔出血症灭活冻干疫苗的免疫效果，以期为硒的研究积累基础资料，同时为硒在养兔业上的应用提供理论依据。 1、不同浓度硒源对兔体外淋巴细胞转化功能的影响 将3种硒源配制成不同的浓度，分别添加到淋巴细胞培养液中，测定各种硒源刺激淋巴细胞培养24h、48h的转化率，发现3种硒源在24h对细胞有很好的刺激作用，其中亚硒酸钠在1×10^-8、5×10^-8Mol/L，硒化卡拉胶在1×10^-7Mol/L，硒代蛋氨酸在1×10^-6、5×10^-6Mol/L可显著促进PHA对淋巴细胞的刺激作用。另外比较了3种硒源对来自同一个体淋巴细胞刺激24h的作用差异，结果显示，亚硒酸钠、硒化卡拉胶分别在1×10^-7Mol/L、1×10^-6Mol/L浓度显著促进PHA刺激的淋巴细胞转化，但两者在1×10^-5Mol/L对淋巴细胞转化有明显抑制；硒代蛋氨酸则从1×10^-10到1×10^-5Mol/L的浓度范围内对细胞均有促刺激作用，浓度越大刺激越明显，1×10^-5Mol/L效果最好。 2、不同硒源对药物抑制兔淋巴细胞转化逆转作用的比较 选用氢化可的松和环磷酰胺建立两种淋巴细胞转化的免疫抑制模型，分别将3种硒源同时或先于抑制药物3h加入细胞培养液中，24h后用MTT法测定细胞转化值，比较其逆转效果。结果显示，两组药物抑制实验中，预先添加各种硒源除1×10^-5Mol/L浓度的亚硒酸钠和硒化卡拉胶外，其余均可有效地保护细胞抵抗药物的抑制作用，使细胞转化恢复至正常水平，其中硒代蛋氨酸效果最好，适量的硒代蛋氨酸不仅可以逆转受抑细胞，且与正常对照比较还有明显的促进作用。硒和氢化可的松一同加入培养时，适量的硒代蛋氨酸和亚硒酸钠可逆转受抑细胞，使其转化恢复到正常水平，其中以硒代蛋氨酸效果较好，但硒化卡拉胶对受抑细胞没有逆转作用。硒和环磷酰胺一同加入培养时，3种硒源除1×10^-5Mol/L浓度的亚硒酸钠和硒化卡拉胶外，其余均可有效的逆转受抑细胞，其中以硒代蛋氨酸的作用最好。不同硒源对兔免疫功能和抗氧化能力形响的研究3、不同硒源对兔体液免疫和杭氧化能力的影响 实验兔”只，均分7组，在接种兔出血症灭活冻干疫苗的同时，将3种硒源按0.lmg/kg和0.3mg/kg两种剂童注射机体，另设生理盐水对照，分别于免疫前1d、免疫后1 Od、ZOd、3Od无菌心脏采血，测定血清杭体水平、GSH一Px活力和MDA含童.结果显示，在杭体产生方面，免疚后10d，硒化卡拉膨0.3mg/kg)组、亚硒酸钠（0 .lmg/kg）组和硒代蛋氮酸（0 .3mg/kg）组杭体水平显著提高;20d、30d，硒化卡拉胶（0 .3mg/kg）组和硒代蛋氨酸（0 .3mg/kg）组杭体水平显著提高.在GSH一Px活力方面，10d，亚硒酸钠（0.3mg/kg）组酶活力最高;20d，各注射硒组酶活力均有提高，其中亚硒酸钠（0.lmg/kg和0.3mg/kg）组、硒化卡拉胶（0.zmg/kg和0.3mg/kg）组显著提高;30d，亚硒酸钠（0 .3mg/kg）组、硒化卡拉胶（0 .3mg/kg）组酶活力仍保持较高水平.各注射硒组均可降低血清MDA含量，以硒化卡拉胶（0 .3mg/kg）组效果最好.4、不同住剂对兔出血症灭活冻干疫苗免疫效果的影响 健康新西兰青年兔20只，随机分成4组，在接种兔出血症灭活冻干苗的同时，各组分别注射左旋咪吐（LMS，2 mg/kg），黄茂多糖（^PS，2 .4 mg/kg），亚硒酸酸钠（Se，0.1 mg/kg）和生理盐水，于免疫后第8、14、21、28天无菌，心脏采血，应用间接ELISA试验和MTT比色法检测机体杭体水平和淋巴细胞转化功能.结果显示，晰S组抗体和淋巴细胞转化水平在1周后即可明显上升，与对照比较差异显著（P<0 .05），但持续时间不长;APS组，试验期内杭体水平一直处于较高水平（P< 0.05），且能有效刺激淋巴细胞转化;se组，刺激抗体产生和淋巴细胞转化方面效果比LMS好，但作用时间不如APS长.更多还原

【Abstract】 Many researchers reported that the immune function of poultry, livestock and human could be damaged oweing to deficiency of selenium. While to supply animals with proper selenium could strengthen the response of cell immunity, humour immunity and non-specific immunity. But till now, few researches were seen about the effect of selenium on the immune function of rabbit. And few were done at one time with different selenium resources on the immunity of animals. The purpose of this study was to compare the effect of different selenium resources, sodium selenite, Kappa-selenocanageenan and DL-selenomethionine on the immune function and anti-oxidation of rabbit. In addition, the immune effects of sodium selenite, levamisole and Astragalus polysachain on the RHD freeze-dried vaccine were studied.1% Effects of different selenium resources on rabbit lymphocyte transformation stimulated by PHA in vitro. Different concentrations of selenium from 1 10-10MoL/L to lxlO"5MoL/L were added to the rabbit lymphocyte culture stimulated by PHA in vitro. Lymphocyte transformation was examined respectively at 24h, 48h with MTT colorimetric assay. Results indicated that at 24h, sodium selenite, Kappa-selenocanageenan and DL-selenomethionine could markedly stimulate the lymphocytes transformation with PHA at l 10-8MoL/L and 5 10-8MoL/L, l 10-7MoL/L , 1 10-6MoL/L and 5 10-6MoL/L (P<0.05) respectively. While at 24h, working on the lymphocyte from the same rabbit, DL-selenomethionine exhibited the best effect. It could promote the lymphocytes transformation at all the concentrations from l 10-10MoL/L and l 10-5MoL/L (P<0.05) , especially at l 10-5MoL/L (P<0.01). While Sodium selenite and Kappa-Selenocanageenan just at that of 1 10-7MoL/L and 1 10-6MoL/L ( P<0.05 ) , both exhibited inhibitory function at l 10-5MoL/L (P<0.05) .2, The reversion of different selenium resources on lymphocyte transformation restrained by inhibitor. We selected hydrocortisone and cyclophosphamide to build the model of immunosuppression in lymphocyte culture, while selenium added to the culture simultaneously with inhibitors or 3h before them. Lymphocyte transformation was examined at 24h with MTT colorimetric assay. The results revealed that pre-added selenium could effectively protect lymphocytes to resist and reverse the action of inhibitorsat most concentrations except l 10-5MoL/L of sodium selenite and Kappa-selenocanageenan. Among them, DL-selenomethionine showed the best, which could even promote lymphocyte transformation at proper concentration. When added simultaneously with hydrocortisone, DL-selenomethionine and sodium selenite at proper concentration could reverse the depressed lymphocyte transformation to the normal level, but Kappa-selenocanageenan had not the effect. When added simultaneously with cyclophosphamide, except 1 10-5MoL/L of Sodium selenite, Kappa-selenocanageenan, all other concentrations exhibited good effect, DL-selenomethionine was the best. 3% Effects of different selenium resources on the function of rabbit humour immunity and anti-oxidation in vivo. Thirty-five rabbits were randomly divided into seven groups and vaccinated with RHD freeze-dried vaccine. At same time, the rabbits were injected respectively with sodium selenite (0.1 mg/kg and 0.3 mg/kg) , Kappa-selenocanageenan (0.1 mg/kg and 0.3 mg/kg) , DL-selenomethionine (0.1 mg/kg and 0.3 mg/kg) and physiological salt solution as control. Antibody against RHD, activity of GSH-Px and content of MDA in rabbit serum were detected on 0, 10, 20, 30d after inoculation. Results showed that sodium selenite (0.1 mg/kg) , Kappa-selenocanageenan (0.3 mg/kg) , DL-selenomethionine (0.3 mg/kg) could increase the level of RHD antibody, while sodium selenite (0.1 mg/kg and 0.3 mg/kg) , Kappa-selenocanageenan (0.1 mg/kg and 0.3 mg/kg) improved the activity of GSH-Px. Kappa-selenocanageenan (0.3 mg/kg) , whereas, represented good effect on eliminating the MDA in serum.4, Effects of different adjuvants of RHD freeze-dried vaccine on the immune function of rabbits. Twenty rabbits were r更多还原