【作者】 陆扬；

【导师】 朱伟云；

【作者基本信息】 南京农业大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 本论文试验部分介绍了3个动物试验,涉及机体生命过程中3个重要时期(发育、过敏、感染)胃肠道的健康。前两个实验主要研究了大豆苷元对胃肠道发育和免疫应答以及微生物区系的影响。在第一个动物试验中,以断奶仔猪为观察对象,研究了大豆苷元对断奶前后胃肠道组织的发育,并发现大豆苷元对断奶仔猪胃壁组织的肥大细胞有显著抑制作用；此后,文章的第二个动物试验以OVA致敏小鼠,建立过敏模型,进一步研究其对肥大细胞的影响,两个试验虽然将大豆苷元的功能与肥大细胞联系在了一起,但其作用机理还有待进一步研究。文章的第三个动物试验侧重于肠道免疫学基础研究,涉及了目前免疫学研究热点TLRs。该动物试验对一株有毒力的产气荚膜梭菌感染后,各种TLR及其相关分子的表达进行了研究,并发现肠道TLR的作用方式可能与脾脏不同,为以后促进肠道健康研究提供理论基础。1.大豆苷元对断奶仔猪胃肠道消化道发育的影响本试验研究了哺乳期饲喂大豆苷元对仔猪胃肠道组织发育的影响。3窝新生仔猪,在窝内随机分为两组：大豆苷元组和对照组,于7、9、11日龄每头仔猪分别饲喂1、2、3mg大豆苷元-脱脂乳溶液或脱脂乳安慰剂；所有仔猪于21日龄断奶；14、21、24和35日龄,每窝随机抽取饲喂大豆苷元和对照组仔猪各一头屠宰,取胃底部、十二指肠前端、空肠前端进行组织学观察。结果显示,大豆苷元处理组胃底腺盐酸细胞显著多于对照组(P<0.05)；断奶造成仔猪十二指肠和空肠肠绒毛高度变短,胃底腺增厚速度变快,但大豆苷元对肠绒毛高度和胃底腺厚度都无显著影响,结果说明,哺乳期饲喂大豆苷元可能对断奶仔猪胃底盐酸细胞发育有促进作用。2.大豆苷元对断奶仔猪胃肠道免疫应答的影响本试验研究了哺乳期饲喂大豆苷元对断奶仔猪胃肠道免疫器官重量以及免疫应答的影响。3窝新生仔猪,在窝内随机分为两组：大豆苷元组和对照组,于7、9、11日龄每头仔猪分别饲喂1mg、2mg、3mg大豆苷元-脱脂乳溶液或脱脂乳安慰剂；所有仔猪于21日龄断奶；14、21、24和35日龄,每窝随机抽取饲喂大豆苷元和脱脂乳仔猪各一头称重、屠宰、称量脾脏和胸腺重；取胃底部制成组织切片显微观察并统计胃底腺肥大细胞数量；测定空肠和回肠组织组胺以及血清皮质醇。结果显示,对照组仔猪脾脏和胸腺重量在断奶第2周重显著下降(P<0.05),大豆苷元组胸腺重在断奶后第2周显著大于对照组(P<0.05),脾脏和胸腺重在断奶前后无显著变化(P>0.05);断奶可短暂引起仔猪胃组织中肥大细胞大量增加,大豆苷元组肥大细胞在断奶前和断奶后第3天都显著少于对照组(P<0.05);可能由于错过了最佳观察时间,仔猪断奶后第3天和第2周都未见空肠和回肠组胺显著升高,但断奶后大豆苷元组回肠组胺含量显著低于对照组(P<0.05)。结果说明,哺乳期饲喂大豆苷元可以抑制断奶造成的胸腺萎缩和肥大细胞大量增殖,减少断奶仔猪回肠组胺合成。3.大豆苷元对食物过敏小鼠免疫应答的影响本试验利用OVA诱导小鼠食物过敏,研究大豆苷元和染料木素对其血清OVA特异性抗体水平和肥大细胞数量的影响。结果显示,致敏组小鼠血清OVA-IgG1滴度剧烈增加,肥大细胞虽没有显著变化(P>0.05),但mMCP-7阳性肥大细胞数量显著(P<0.05)。口服大豆苷元和染料木素显著降低OVA-IgG1水平,减少了肥大细胞数量,尤其是mMCP-7阳性肥大细胞的数量显著降低(P<0.05)；染料木素对染料木素的抑制作用显著强于大豆苷元(P<0.05),结果说明大豆苷元和染料木素可缓解食物过敏的发生进程。4.大豆苷元对食物过敏小鼠盲肠微生物区系的影响本试验利用PCR/DGGE技术研究了OVA诱导过敏对小鼠盲肠食糜微生物区系的影响,以及大豆苷元和染料木素对致敏小鼠盲肠食糜微生物区系的影响。结果表明,OVA诱导过敏和大豆苷元或染料木素对小鼠盲肠食糜的DGGE条带数和多样性指数皆没有显著影响。DGGE图谱显示,OVA诱导导致一特导条带消失,但口服大豆苷元或染料木素的致敏小鼠仍可观察到该特征条带存在；切胶测序后,基因序列比对结果显示,该特征条带所对应的微生物与Atopostipes suicloacalis的相似性为96%。口服大豆苷元或染料木素小鼠导致某一特异条带出现,切胶测序后,基因序列比对结果显示,该特征条带所对应的微生物与Clostridium metallolevans strain ASI1相似性为99%。结果说明,OVA过敏诱导没有导致Balb/c小鼠盲肠微生物区系优势条带数减少或多样性降低,但使个别细菌的优势种群地位消失,口服大豆苷元或染料木素可稳定这些优势菌群,大豆苷元和染料木素可使某些菌群得以富集。5. TLRs在产气荚膜梭菌感染进程中的表达本试验利用分子生物学方法研究肉鸡在产气荚膜梭菌感染后TLR及其相关配体和细胞因子的表达。结果表明,产气荚膜梭菌攻毒后,脾脏和回肠中TLR及其相关配体和细胞因子变化规律明显不同：在脾脏中,TLR1.2、TLR2.1和TLR15显著上调,且彼此高度相关(P<0.05),但其部分相关配体和细胞因子出现下调或有下调趋势；在回肠中,TLR1.1、TLR2.2和TLR21在攻毒后上调,且彼此高度相关(P<0.05),其相关分子MyD88、TRAF6、TRIF、IRF3、RIP-1和IL-8的表达都在攻毒后显著上调(P<0.05)；回肠类TNF-α的表达也显著上调(P<0.05),但与各TLRs无显著相关性。此外,TLR4和TLR7在脾脏中的表达也有所上调,但在回肠中无显著变化。结果说明,TLR2亚群在产气荚膜梭菌感染中起重要作用,但其表达模式在回肠和脾脏中存在很大差异,脾脏的免疫应答在感染后可能存在抑制现象,TLR1.2/TLR2.1/TLR15可能是TLR2亚群在脾脏的主要结合模式；回肠IL-8显著升高,说明免疫应答被活化,TLR1.1/TLR2.2/LR21可能是TLR2亚群在回肠的主要结合模式,该配对在起始免疫活化时可能有MyD88、TRAF6、TRIF、IRF3、RIP-1的参与。然而,回肠类TNF-α的升高可能与产气荚膜梭菌致病机理有关。更多还原

【Abstract】 Three animal trials were conducted in this study which involved the gastrointestinal health status during developmental, allergic or infection phases of the animal. The first two trials investigated the effects of daidzein on the development, immune response and microflora in gastrointestinal tract of animals. In the first trial, obvious inhibitory effect was observed on the mast cells of weaning piglets, thus, in the second trial, an allergic mice model was established by using OVA and used to investigate the relation between daidzein and mast cells. In the third animal trial, TLRs signal pathway during Clostridium perfringens infection was investigated and different cooperation model of TLR2 subfamily was found which may function in ileum and spleen. This study could provide theoretical basis to future research for how to promote intestinal health.1. Effects of daidzein on the development of gastro-intestinal digestive system of weaning pigletsNeonatal piglets from three litters were randomly divided into two groups within each litter. That was half of the piglets from each letter were fed with 1,2 and 3 mg of daidzein-skimmed milk at 7,9 and 11 days of age, respectively, and other half of the piglets were fed with the same volume of skimmed milk as control. All piglets were weaned at 21 days of age. At 14,21,24 and 35 days of age, within each litter, one piglet from each group were weighed and slaughtered. Tissues of stomach bottom, proximal parts of duodenum and jejunum were collected for histological analysis. The results showed that daidzein increased the number of the oxyntic cells in fundic gland significantly (P<0.05). No effect of daidzein was observed on the thickness of fundic gland or intestinal villus height (P>0.1).2. Effects of daidzein on the immune system in gastro-intestine of weaning piglets Neonatal piglets from three litters were randomly divided into two groups within each litter. That was half of the piglets from each letter were fed with 1 mg,2 mg and 3 mg of daidzein-skimmed milk at 7,9 and 11 days of age, respectively, and other half of the piglets were fed with the same volume of skimmed milk as control. All piglets were weaned at 21 days of age. At 14,21,24 and 35 days of age, within each litter, one piglet from each group were weighed and slaughtered, and spleen and thymus were weighed, tissues of stomach bottom collected for histological analysis, tissues of jejunum and ileum collected for histamine measurement, and serum collected for cortisol measurement. The results showed an inhibitory effect of daidzein on the accumulation of mast cells in the fundic gland which was caused by weaning. Moreover, inhibitory effect of daidzein was observed on the histamine synthesis in the ileum at three days and two weeks post weaning (P<0.05), although no significant increase of histamine levels in intestinal tissue was observed after weaning. A decrease in weight of thymus and spleen was observed in control group but not in daidzein group, at two weeks post weaning, especially the thymus of daidzein group was heavier than that of control group at two weeks post weaning (P<0.05). In conclusion, daidzein administeration during lactation can inhibit the atrophy of thymus gland and mastocytosis caused by weaning, and decrease histamine synthesis in ileum after weaning.3. Effects of daidzein and genistein on the immune response of food allergic miceThe food allergy in mice was induced by using OVA and the induced allergic mice was then used to investigate the effects of daidzein and genistein on the antibody level and mast cell of the food allergic mice. The results showed that the OVA-IgGl, the number of mMCP-7 positive cells increased in the serum of sensitized mice dramatically (P<0.05). Inhibitory effects of daidzein and genistein were observed on the OVA-IgG1 and the number of mall cells, especially on the mMCP-7 positive cells (P<0.05). The inhibitory effect of geistein is stronger than that of daidzein (P<0.05).4. Effects of daidzein on the caecum microflora of food allergic miceThe effects of OVA challenge, daidzein and genistein treatment on the caecum microflora community of OVA-sensitized mice were further investigated. DNA was isolated from the caecum digesta of the trial mice for the PCR/DGGE analysis using the primers targeting V6-V8 area of 16S rDNA of bacteria. The diversity of intestinal microbial populations of the mice was determined based on the shannon’s index. And the interesting bands on DGGE gels were cut, sequenced and compared with the sequences of GenBank. Result showed no significant different in number of DGGE bands or shannon’s index between OVA challenge, daidzein or genistein treatments. However, OVA challenge caused the disappearance of one specific band (named as m-b). In samples from mice with oral administration of daidzein or genistein, this band remained present. Sequence analysis showed the the closest related sequence to band m-b in GenBank is Atopostipes suicloacalis strain PPC79, with 96% similarity. A novel band named m-a was observed in the daidzein and genistein treatment group, which is closely related to Clostridium metallolevans strain ASI1, with 99% similarity. In conclusion, food allergy did not affect the general diversity of the intestinal microbe community, but caused the disappearance of certain dominant bacteria species, but this species remained with daidzein or genistein administration. The results suggest that daidzein and genitein are likely to enrich certain bacteria species which may take part in the metabolism of daidzein and genistein.5. Gene expression profiles of toll-like receptor-mediated signaling during clostridium perfringens infectionToll-like receptors (TLRs) participate in detecting microbial pattern molecules for activation of host immune response. We investigated possible roles of TLRs in the chicken response to Clostridium perfringens infection by examining the expression of TLRs and other genes involved in TLR-mediated signaling within the spleen and ileum of C. perfringens challenged chickens. The members of TLR2 subfamily were found to be involved in the host response. A combination of TLR1.2, TLR2.1 and TLR15 appeared to play a major role in the splenic response, following no significant TLR associate adaptors or interleukins up-regulated and down-regrulated of TNF-a at D4 PI. A combination TLR1.1, TLR2.2 and TLR21 appeared to play a major role in the spleenic response and increased thre expression of IL-8 through MyD88, TRAF6, TRIF and RIP-1 in the ileum. However up-regulation of TNF-αwas observed in the ileum of the challenged chicken, which had no relation with TLR2 but seemed to take part in the pathogenesis of C. perfringens. Besides, TLR4 and TLR7 were up-regulated in the spleen of the challenged chicken.更多还原