【作者】 赖长华；

【导师】 李德发； 尹靖东；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2005， 博士

【摘要】 本论文通过两个动物试验及一个体外细胞培养实验,系统地探讨了共轭亚油酸（CLA）对免疫应激仔猪免疫功能的调控作用及其作用机理。（1）初步探讨了CLA对早期断奶仔猪生长性能及免疫反应的影响,并确定适宜的CLA添加剂量。试验选用72头23±2 d断奶仔猪,随机分为4个处理,每个处理6个重复,每个重复3头猪,分别饲喂添加0%、1%、2%和3%CLA的日粮。试验期28 d。试验结果显示,随CLA添加剂量的增加,日增重和日采食量呈二次曲线变化,饲料转化效率线性改善（P<0.05）;特异的OVA抗体水平呈二次曲线变化（P<0.05）;外周血淋巴细胞转化率呈线性提高（P<0.05）。综合生长性能与免疫反应考虑,CLA的最佳添加剂量为2%。（2）通过给断奶仔猪注射脂多糖（LPS）以模拟免疫应激状态,研究CLA缓解免疫应激引起的仔猪生长抑制及对免疫反应的调控作用,并利用分子生物学手段探讨了CLA缓解免疫应激的作用机理。试验选用72头28±2 d断奶仔猪,采用2×2因子试验设计:日粮类型（2%CLA或2%大豆油）和免疫应激（注射LPS或生理盐水）。试验期28 d。在试验第14和21 d,每日粮处理中一半的仔猪注射100 pg/l(g体重的LPS,另一半注射等量的生理盐水。注射后3 h和24 h前腔静脉采血,并在第21 d培养细胞提取核蛋白。在试验第29 d,注射LPS或生理盐水后3 h屠宰仔猪取脾脏和胸腺,测定前炎性细胞因子和过氧化物酶体增殖物激活受体-γ（PPARγ）的mRNA相对含量。试验结果表明,CLA缓解了LPS刺激引起的日增重降低,改善了饲料转化效率（P<0.05）。同时,CLA提高了外周血淋巴细胞转化率和CD8⁺淋巴细胞亚群的百分含量（P<0.01）,并降低了CD4⁺/CD8⁺比率（P<0.05）。但是,CLA抑制了（P<0.05）因注射LPS引起的血浆白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）、急相期蛋白（α-AGP）和PGE₂含量的升高,CLA也降低了（P<0.05）脾脏和胸腺IL-1β、IL-6和TNF-ct mRNA相对含量,但增强了PPARγ的活性及其mRNA丰度,提高了PPARγ内源配体15d-PGJ₂的含量（P<0.05）。这表明CLA缓解免疫应激引起的生长抑制不是通过抑制免疫系统的活性实现的,而是通过调节PPARγ的活性及其mRNA表达,从基因表达水平调控炎性细胞因子的产生实现的。（3）为确定CLA两种主要异构体在缓解免疫应激及调节免疫反应中的作用,实验用含不同浓度（0、50、100、150和200μM）的c9,t11-CLA、t10,c12-CLA及其1:1混和物的RPMI-1640细胞培养液培养断奶仔猪外周血淋巴细胞,以亚油酸（LA）为对照,研究c9,t11-CLA和t10,c12-CLA对仔猪外周血淋巴细胞转化率的影响。同时用含100 μmo1/L上述各脂肪酸的培养液培养淋巴细胞,提取细胞核蛋白和总RNA,测定PPARγ活性与IL-1β、IL-6、TNF-α和PPARγ mRNA丰度。结果表明,低浓度的c9,t11-CLA和t10,c12-CLA增强了淋巴细胞的转化,高浓度时抑制了淋巴细胞转化,两种CLA异构体都抑制了IL-1β、IL-6和TNF-α的产生和表达,增强了PPARγ的活性和mRNA表达。但在调节淋巴细胞转化和抑制前炎性细胞因子的表达方面,t10,c12-CLA的作用比c9,t11-CLA强。而且,CLA的两种主要活性异构体对仔猪淋巴细胞功能的调控作用是通过调节核受体PPARγ,的基因表达及活性而实现的。更多还原

【Abstract】 Two animal feeding experiments and one in vitro experiment were conducted to investigate the immunomodulation and action mechanism of conjugated linoleic acid （CLA） on immune function of pigs in immunological stress. （1） To explore the effects of CLA on growth and immune response in early weaned pigs and the optimal dose of dietary CLA, 72 cross-bred pigs weaned at age of 23 ± 2 d were randomly allotted to feed one of four diets containing 0, 1, 2, or 3% CLA for 28 days. There were six replicates per treatment with three pigs each pen. With increased dietary CLA, average daily gain and average daily feed intake were increased quadratically （P < 0.05） and feed efficiency was increased linearly （P < 0.05）; Specific ovalbumin antibody was increased quadratically （P < 0.05）, and lymphocyte proliferation was enhanced linearly （P < 0.05）. For improving growth and enhancing immune response, 2% CLA is optimal in weaned pigs. （2） An immunological stress model was establish through intraperitoneal injecting with E. coli lipopolysaccharide （LPS） to further evaluate the effects of CLA on growth-depression induced by immune challenge and immune response of pigs in immunological stress. 72 weaned pigs at 28 ± 2 d of age were used in a 2 2 factorial design. The main factors consisted of diets （2% CLA or 2% soybean oil） and immunological challenge （LPS or saline）. On d 14 and 21, half of pigs in each dietary treatment were injected intraperitoneally with either 100 ug/kg BW LPS or an equivalent amount of sterile saline. Blood samples were collected at 3 and 24 h post-injection and lymphocytes were cultured to extract nuclear protein on d 21. Pigs were killed at 3 h post-challenge on d 29. Spleen and thymus were removed immediately for total RNA extraction to determine the mRNA relative contents of proinflammatory cytokines and peroxisome proliferator-activated receptor- （PPARy）. CLA prevented the growth-suppression due to LPS challenge and improved feed efficiency （P < 0.05）; CLA increased lymphocyte proliferation and percentage of CD8⁺ lymphocyte subsets （P < 0.01） and decreased the ratio of CD4+/CD8+ （P < 0.05） at 24 h post-injection. However, CLA decreased （P < 0.05） the production of plasma interleukin-6 （IL-6）, tumour necrosis factor-a （TNF-a）, acute phase protein （a-AGP） and PGE₂ induced by LPS challenge; CLA dereased the mRNA abundance of IL-1, IL-6 and TNF-a in spleen and thymus, while increased the mRNA abundance and activation of PPARy and the content of 15d-PGJ2 （P < 0.05）. The results indicated that CLA alleviating growth depression after immune challenge is not dependent on suppressing immune system reactivity, but modulating the pro-inflammatory cytokines production at molecular level via regulating the PPARy expression and activation. （3） In order to determine the effects of two CLA isomers on anti-immunological stress and regulating immune response, peripheral blood lymphocytes of weaned pigs were cultured with media containing various concentrations （0, 50, 100, 150 and 200 umol/L） of c9, til-CLA, t10, cl2-CLA or CLA mixture, with linoleic acid （LA） as control, to investigate the effect of CLA isomers on peripheral blood lymphocyte proliferation. In addition, lymphocytes were cultured with media containing 100 umol/L fatty acids as described above and nuclear protein and total RNA were extracted to test PPARy activation and the mRNA relative contents of IL-lp\ IL-6, TNF andPPAR. Results suggested that c9, tll-CLA and tlO, cl2-CLA enhanced lymphocyte proliferation at low concentration, while inhibited lymphocyte proliferation at high concentration. Two CLA isomers suppressed the production and mRNA expression of IL-l\ IL-6 and TNF-a, whereas enhanced the activation and gene expression of PPARy in lymphocyte. The effects of t10, C12-CLA isomer on modulation of lymphocyte proliferation and inhibiting the mRNA expression of proinflammatory cytokines were greater than those of c9, t11-CLA isomer. Moreover, c9, tll-CLA and tlO, C12-CLA regulates immune function of lymphocyte in weaned pigs by modulating the PPARy act更多还原