【作者】 李思明；

【导师】 周定刚；

【作者基本信息】 四川农业大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 防御素是家禽先天免疫的重要效应分子,在宿主抵御致病性微生物入侵时起至关重要的作用。维生素D₃（VD₃）作为一种免疫调节剂,能直接诱导哺乳动物单核细胞、上皮细胞等细胞系中抗菌肽和相关免疫因子的表达。然而,家禽体内尚未发现VD₃转化成1,25（OH）₂D₃活性形式的1α羟化酶。本研究旨在探讨VD₃与鸡体内β-防御素表达之间的关系,同时也探讨了VD₃对丝毛鸡生产免疫性能的影响,共包括以下六部分试验。试验一注射VD₃对丝毛鸡β-防御素表达的影响本试验旨在研究腹腔注射VD₃对丝毛乌骨鸡组织中β-防御素（β-defensins,AVBDs）、Toll样受体（Toll-like receptors,TLRs）、维生素D受体（vitamin D receptor,VDR）和24羟化酶（CYP24）基因表达的影响。采用实时荧光定量PCR方法检测VD₃对6周龄丝毛乌鸡肝、肾、脾、十二指肠、空肠、回肠、盲肠和法氏囊中AVBDs、TLRs、VDR和CYP24基因的表达变化。结果表明,在注射VD₃后24h内,6个AVBDs（AVBD-1、AVBD-5、AVBD-7、AVBD-9、AVBD-10和AVBD-12）、3个TLRs（TLR2、TLR6和TLR 7）、VDR和CYP24基因在肠道组织中均有显著的表达上调;TLR2、TLR5、VDR、AVBD-6、CYP24和GAL-1基因分别在肝、肾、脾组织也有显著的表达变化。结果提示,AVBDs、TLRs、VDR和CYP24基因的表达变化可能是受VD₃的诱导。试验二注射沙门氏菌对丝毛鸡β-防御素基因表达的影响本试验旨在研究腹腔注射灭活沙门氏菌对丝毛乌骨鸡组织中AVBDs和TLRs基因表达的影响。采用实时荧光定量PCR方法检测沙门氏菌对6周龄丝毛乌鸡胸腺、肝、肾、脾、十二指肠、空肠、回肠、盲肠和法氏囊中AVBDs和TLRs基因的表达变化。结果表明,在注射后32h内的不同时间点,8个AVBDs（AVBD-1、GAL-1、AVBD-5、AVBD-6、AVBD-7、AVBD-9、AVBD-10和AVBD-12）和5个TLRs（TLR1、TLR4、TLR5、TLR6和TLR7）基因在丝毛乌鸡胸腺、肝、肾、脾、十二指肠、空肠、回肠、盲肠和法氏囊中均有显著的表达上调。结果提示,在这9个组织中的8个AVBDs和5个TLRs基因的表达发生了大幅上调,AVBDs的表达可能是TLRs的介导所致。试验三饲喂VD₃对丝毛鸡β-防御素基因表达的影响本试验旨在研究VD₃饲喂剂量对丝毛乌骨鸡组织中AVBDs、TLRs、维生素D羟化酶结合蛋白（VDHA）和24羟化酶（CYP24）基因表达的影响。1—6周龄分别饲喂每千克饲料添加0、800、1600、3200和6400IU VD₃的饲粮。采用实时荧光定量PCR方法检测VD₃对6周龄丝毛乌鸡胸腺、肝、肾、脾、十二指肠、空肠、回肠、盲肠和法氏囊中AVBDs、TLRs、VDHA和CYP24基因的表达变化。结果表明,在饲喂800、1600、3200和6400IU VD₃的不同处理中,6个AVBDs（AVBD-1、AVBD-5、AVBD-7、AVBD-9、AVBD-10和AVBD-12）基因在胸腺、十二指肠、空肠、回肠、盲肠和法氏囊组织中的表达均有显著的影响;TLR2、TLR5、VDHA、AVBD-6、CYP24和GAL-1基因分别在肝肾脾组织也有显著的表达变化。结果提示,AVBDs、TLRs、VDHA和CYP24基因的表达变化是受VD₃的诱导,且AVBDs的表达可能受TLRs、VDHA和CYP24基因的介导,其表达与VD₃存在剂量依赖性,适宜的VD₃饲喂剂量可以使β-防御素基因的表达上调。试验四饲喂VD₃对丝毛鸡组织中免疫因子的影响本试验旨在研究VD₃对丝毛鸡体内细胞因子IL-2和IL-18表达的影响,探讨其与细胞因子表达丰度的关系。采用VD₃饲喂模型,利用实时荧光定量PCR技术检测细胞因子在胸腺、肠道和法氏囊中的表达变化。结果表明,长期饲喂6400IU的VD₃有助于IL-18基因在丝毛鸡胸腺、法氏囊和肠道的表达上调。饲喂高剂量也有助于IL-2在十二指肠、法氏囊和盲肠的表达上调,但在胸腺、空肠和回肠中高剂量对其有抑制作用。结果提示,VD₃剂量跟丝毛鸡体内细胞因子IL-2和IL-18表达丰度相关,但其对不同组织中的细胞因子的调节存在差异。试验五饲喂VD₃对丝毛鸡生长性能和免疫器官的影响本试验旨在研究日粮中添加不同水平VD₃对丝毛鸡生长性能和胸腺、脾和法氏囊组织的影响。选用7日龄仔鸡120只,随机分为6个处理,每个处理20羽。试验日粮参照中国地方鸡推荐标准配制,6个处理的日粮VD₃水平分别为:0、400、800、1600、3200和6400 IU/kg。研究结果表明:在本试验范围内,增加日粮VD₃水平可以改善丝毛鸡日增重和平均体重。随着日粮VD₃水平的增加,脾脏和法氏囊组织的质量指数和细胞数随之提高,但不影响胸腺组织的质量指数和细胞数。由此可见,在试验范围内,提高日粮VD₃水平可以显著提高丝毛鸡生长性能,显著增加脾脏和法氏囊组织的脾脏指数和法氏囊指数及其细胞数。试验六丝毛鸡β-防御素的组织表达分布及其基因片段的克隆与序列分析用设计的特异性引物,运用Real-time PCR方法分析了丝毛鸡6个β-防御素基因在不同组织中的分布,结果发现:AVBD-1、AVBD-5、AVBD-7、AVBD-9、AVBD-10、AVBD-12的表达广泛分布在胸腺、十二指肠、空肠、回肠、盲肠和法氏囊组织中,但表达量有差异。同样采用RT-PCR方法扩增丝毛鸡β-防御素基因AVBD-10。通过克隆、测序获得AVBD-10基因的cDNA核苷酸序列,并提交到GenBank,比较分析其与GenBank中注册的防御素基因核苷酸序列的相似性,其核苷酸的相似性在88%～100%之间。利用DNAStar软件对所获得的丝毛鸡β-防御素AVBD-10基因进行系统发育树分析,结果AVBD-10与GenBank中注册的丝毛鸡防御素AVBD-10基因不在同一分支,可能是品种血缘不纯造成的差异。总之,VD₃能诱导丝毛鸡体内防御素AVBDs的表达,其诱导功能的发挥与TLRs、VDHA和Cyp24的介导有关;适宜的VD₃饲喂剂量能上调组织中AVBDs、IL2、IL18基因的表达,提高生长性能和免疫器官质量指数。更多还原

【Abstract】 Avian beta defensins（AVBDs） are important effector molecules of innate immune,which play a critical role in the host against invasion of pathogenic microorganisms.Vitamin D3（VD3） as an immunomodulator,could directly induce the expression of antimicrobial peptide and related immune factors in mammalian monocytes, epithelial cells,however in poultry,lacking the 1α-hydroxylase of vitamin D3 into the 1,25 dihydroxy-vitamin D3.Thus,they were studied that of the relationship of VD3 and the expression ofβ-defensin between,and the effect of VD3 on the performance of production and immune,including the following six experiments.Exp.1 Effect of Vitamin D3 by Injection on theβ-defensins in Taihe Silky FowlThe expression of AVBDs,Toll-like receptors（TLRs） and vitamin D receptor（VDR） was studied following in vivo by injection vitamin D₃.Healthy 6w Silky Fowl were abdominally injected with vitamin D₃ or untreated.Real-time PCR analyses reveal that injection of vitamin D₃ showed significantly up-regulated the expression of TLRs （TLR2,TLR6 and TLR7）,VDR,AVBDs（AVBD 1,AVBD 5,AVBD 7,AVBD 9,AVBD 10 and AVBD 12） and 24-hydroxylase（CYP24A1） in the gut（duodenum,jejunum,ileum and cecum） during different time from 8 h to 24 h postinjection,similarly the expression of TLRs（TLR2,TLR5）,VDR,AvBDs（AVBD-6,GAL-1） and 24-hydroxylase（CYP24A1） in the tissues（liver,spleen and kidney）.These results suggest that expression of VDR,AVBDs and TLRs seems to be induced by vitamin D₃ and was concluded that the tissues expressing TLRs and VDR responds to vitamin D₃ and in turn upregulate this tissues cellular functions to synthesize AVBDs.Intraperitoneal injection of vitamin D₃ likely resulted in enhanced the expression of AVBDs,TLRs and VDR,which provided insight into factors important to the control of the innate immune response in the chicken.Exp.2 Effect of Salmonella enteritidis by Injection on theβ-defensins in Silky FowlThe expression of AVBDs and TLRs was studied following in vivo challenge with Salmonella enteritidis.Healthy 6w Silky Fowl were abdominally injected with Salmonella enteritidis or untreated.Real-time PCR analyses reveal that Salmonella enteritidis by injection showed significantly up-regulated the expression of eight avianβ-defensins （AVBD-1,GAL-1,AVBD-5,AVBD-6,AVBD-7,AVBD-9,AVBD-10 and AVBD-12） and five TLRs（TLR1,TLR4,TLR5,TLR6 and TLR7）in the tissues（thymus,duodenum, jejunum,ileum,cecum,bursa of fabricius,liver,spleen and kidney）at 0-32 h postinjection.These results suggest that the Silky Fowl expresses at least eight isoforms of avianβ-defensins and five types of TLRs with a greater expression in the 9 tissues. Thus expression ofβ-defensins and TLRs seems to be affected by Salmonella enteritidis, and was concluded that the tissues expressing TLRs responds to Salmonella enteritidis and in turn upregulate cellular functions to synthesize avianβ-defensins.Such expression and functions of TLRs may play an essential role in Silky Fowl innate immunity for host defense.Exp.3 Effects of Different Supplemental Levels of Vitamin D in Diets onβ-defensins in Taihe Silky FowlThe expression of AVBDs,TLRs and vitamin D3 hydroxylase associated protein （VDHA） was studied following in vivo by feeding vitamin D₃.Healthy Silky Fowl were fed vitamin D₃ of 0,800,1600,3200 and 6400 IU/kg feed from 1 to 6w.Real-time PCR analyses reveal that feeding of vitamin D₃ showed significantly changed the expression of AVBDs（AVBD 1,AVBD 5,AVBD 7,AVBD 9,AVBD 10 and AVBD 12） in the thymus, duodenum,jejunum,ileum,cecum and bursa of Fabricius during different dose from 800 to 6400 IU,similarly the expression of TLRs（TLR2,TLR5）,VDHA and AVBDs （AVBD-6,GAL-1） in the tissues（liver,spleen and kidney）.These results suggest that expression of VDHA,AVBDs and TLRs were induced by vitamin D₃ and the genes expression levels existed a dose-dependent in same tissues of birds with fed VD3 of different dose,and the appropriate dose of VD3 can directly up-regulate the gene expression ofβ-defensins in the tissues.Exp.4 Effect of Vitamin D on the Cytokines in Taihe Silky FowlThe aim of this experiment was to research the effects of VD3 on interleukin-2（IL-2） and interleukin-18（IL-18） in Taihe Silky Fowl.Real-time PCR analyses reveal that VD3 by injection showed significantly changed the expression of IL-2 and IL-18 in the tissues （thymus,duodenum,jejunum,ileum,cecum and bursa of fabricius）.Long-term feeding 6400IU of VD3 in diets was up-regulated the expression of IL-18 in thymus,bursa of Fabricius and gut of Silky Fowl.Feeding high-dose VD3 also was up-regulated the expression of IL-2 in the duodenum,the bursa of Fabricius and cecal,but down-regulated the expression in the thymus,jejunum and ileum.The results suggest that doses of vitamin D3 effect the expression change of IL-2 and IL-18 in Silky Fowl,but this cytokines had regulated different in the different tissues.Exp.5 Effects of Different Supplemental Levels of Vitamin D in Diets on Growth performance and Immune Organs in Taihe Silky FowlThe aim of this experiment was to research the effects of different supplemental levels of vitamin D in diets on growth performance and immune organs in Taihe Silky Fowl.One hundred and twenty Taihe Silky Fowl were divided into 6 treatments and each treatments contained 20 Taihe Silky Fowl.Vitamin D levels in the diets of six treatments were 0, 400,800,1600,3200 and 6400 IU/kg respectively.Other nutrient levels all reached the recommended nutrient standard of Chinese chicken.The results showed that daily gain and average weight of Taihe Silky Fowl were improved with the increase of vitamin D levels. The number of cells of the spleen and bursa were improved with the increase of vitamin D levels,while the number of cells of thymus were not affected.In conclusion,the growth performance of Taihe Silky Fowl were improved with the increase of vitamin D levels,and the the number of cells of the spleen and bursa were improved significantly at the same time.Exp.6 Cloning,Sequencing and Distribution ofβ-defensins Gene in Silky FowlWith specific primers,six chichenβ-defensin cDNA fragments,were analyzed by real-time PCR from Silky Fowl.Different mRNA distribution of genes in different tissues, indicated that AVBD-1,AVBD-5,AVBD-7,AVBD-9,AVBD-10 and AVBD-12 were widely expressed in thymus,duodenum,jejunum,ileum,caecum and bursal tissue,but there were difference in expression.AVBD-10 was amplified by reverse transcription-polymerase chain reaction（RT-PCR）.After being sequenced,AVBD-10 cDNA fragments were identified and deposited to GenBank TM.The results of sequence comparison with theβ-defensin gene sequences published in GenBank TM indicated that nucleotide similarity of the AVBD-10 genes ranged from 88%to 100%.Analysis by DNAstar showed that Silky Fowl AVBD-10 by registered of the GenBank and sequenced AVBD-10 gene were not the same embranchment,which were probably caused by hybrid of species. In summary,VD3,whose function was mediated by TLRs,VDHA and Cyp24, induced the expression of defensins in Silky Fowl;the gene expression of AVBDs,IL2 and IL18 in tissue was up-regulated,and the growth performance and mass index of immune organ were improved with appropriate dose of vitamin D3更多还原