【作者】 曹力；

【导师】 姚军虎；

【作者基本信息】 西北农林科技大学 ， 动物营养与饲料科学， 2012， 博士

【摘要】 肠道的屏障功能对于肉鸡肠道的健康是非常重要的。许多的研究兴趣都聚焦于益生菌对宿主健康的维持、疾病的预防或治疗。乳酸杆菌，作为一种益生菌，对肉鸡肠道健康有重要的改善作用。乳酸菌的黏附定植是其发挥益生作用的前提和基础。尽管众多研究显示乳酸菌能够对宿主的肠道发挥有益的作用，但其对肠道屏障功能作用机理尚不明确。本研究拟筛选出高黏附性乳酸杆菌，研究其对肉鸡肠道黏膜屏障保护作用及机理。为益生菌在生产实践中的推广应用提供科学依据。试验一高黏附乳酸杆菌的筛选及其对肠道黏液蛋白2（Muc-2）的影响本试验选用6株乳酸杆菌菌株，筛选出黏附性能高的菌株，并进行耐酸、耐胆盐的检测，结果显示，Lactobacillus fermentum1.2029显示出高的黏附性，并能够在低pH值及胆盐存在的条件下存活。为了研究其对肠道黏液蛋白表达的影响，将24只1日龄AA肉仔鸡随机分为2个处理组：对照组和乳酸杆菌处理组。处理组每天口腔灌服1ml浓度为108CFU/ml发酵乳酸杆菌，对照组每天口腔灌服1ml的生理盐水。该结果显示，与对照组相比，灌服发酵乳酸杆菌组显著增加了空肠杯状细胞的密度（P <0.05），但对其它肠段的杯状细胞密度没有影响。灌服乳酸杆菌组显著提高了空肠和回肠MUC2的mRNA表达量（P <0.05），分别增加了3.89倍和3.32倍，而十二指肠mRNA也呈增加趋势，但并未达到显著水平。本研究表明，L. fermentum1.2029通过刺激MUC2的表达来保护肉鸡小肠黏膜。试验二Lactobacillus fermentum1.2029对坏死性肠炎肉仔鸡肠道黏膜免疫功能的影响本试验研究了坏死性肠炎条件下，L. fermentum1.2029对肠道黏膜免疫功能的影响，将240只1日龄AA肉鸡被分成3个试验组：对照组，坏死性肠炎（NE）模型组及乳酸杆菌处理组。对照组的鸡只每天口腔灌服PBS，NE模型组鸡只根据模型要求进行产气荚膜梭菌攻毒，乳酸杆菌处理组鸡只在NE模型的基础上每天灌服L. fermentum1.2029（1.0ml/鸡,108CFU/ml）。28日龄，采集样品并进行组织学评分及mRNA分析，同时对肠道进行肉眼评分。结果表明，L. fermentum1.2029降低了NE引起的损伤程度；组织学评分结果显示，对照组肠道黏膜完整无损；NE模型组肠道黏膜样显示了强烈的炎症反应，乳酸杆菌处理组，缓解了炎症症状；肉眼评分结果显示，乳酸杆菌降低了NE引起的损伤程度；Real-time PCR结果显示，与正常对照组相比，NE模型组中，TLR2和IFN-γ的表达分别呈2倍和3倍增加，乳酸杆菌处理后，显著降低了TLR2及IFN-γ的表达（P <0.05）。产气荚膜梭菌感染后，IL-10的表达降低（P <0.05）。各处理组TLR4的表达没有显著的变化。这些结果表明，L. fermentum1.2029通过改变细胞因子及TLR的表达降低了坏死性肠炎的损伤程度。试验三Lactobacillus fermentum1.2029对肠道上皮细胞重建的影响为了研究L. fermentum1.2029对肠道上皮重建的影响，240只1日龄AA肉鸡被分成3个试验组：对照组，坏死性肠炎（NE）模型组及乳酸杆菌处理组。对照组的鸡只每天口腔灌服PBS，NE模型组鸡只根据模型要求进行产气荚膜梭菌攻毒，乳酸杆菌处理组鸡只在NE模型的基础上每天灌服L. fermentum1.2029（1.0ml/鸡,108CFU/ml）。结果显示，L. fermentum1.2029促进了NE肉鸡回肠上皮细胞的迁移（P <0.05），提高了回肠刷状缘膜蔗糖酶的活性及回肠黏膜cdx1的表达（P <0.05）。L. fermentum1.2029处理提高了血液EGF的浓度和回肠黏膜EGFR的表达量（P <0.05），但对隐窝细胞的增殖没有影响。结果表明，L. fermentum1.2029促进了回肠上皮细胞的重建过程，在这一过程中伴随着上皮细胞的分化。试验四Lactobacillus fermentum1.2029对坏死性肠炎肉鸡肠道上皮紧密连接蛋白表达的影响为了探讨L. fermentum1.2029对坏死性肠炎（NE）肉鸡肠道上皮紧密连接蛋白表达的影响，将240只AA肉鸡随机分为3个处理组：对照组，NE模型组及乳酸杆菌处理组。28日龄，每组屠宰24只鸡，采集回肠黏膜样分别用于电镜下形态观察和real-time PCR检测紧密连接蛋白claudin-1和occludin的表达。电子显微镜观察结果显示，乳酸杆菌处理组改善NE模型组的微绒毛结构；Real-time PCR分析显示，乳酸杆菌处理组claudin-1和occludin的表达显著高于NE模型组（P <0.05），但与对照组没有差异。该试验结果表明，L. fermentum1.2029改善了NE肉鸡的微绒毛结构，并能有效上调NE条件下claudin-1和occludin的表达，显示其具有保护肠道屏障功能的作用。更多还原

【Abstract】 Intestinal barrier function is very important for health status of broilers. At present,interest in the use of probiotics for health maintenance and disease prevention or treatmenthas increased markedly. Lactobacillus, as one of probiotics, can improve intestinal health ofbroiler chickens. Adhesion and colonization of Lactobacillus to the intestinal mucosal surfaceare critical prerequisite for exerting beneficial effect to their host. Although many studies haveshown that Lactobacillus exert favorable effects on intestine of the host, the mechanism ofLactobacillus modulating barrier function of small intestine is still not entirely understood. Inthe present experiments, our objective is to select potential hightly adherent strains ofLactobacillus and investigate effects of the strain on intestinal barrier function of broiler, andgain further understanding of the mechanism by which Lactobacillus protect intestinalmucosal barrier. The results of the present experiments will also provide scientific evidencefor application of Lactobacillus in practice.Trial1. Effect of selected Lactobacillus strain with high adhesion on expression ofintestinal mucin-2in broiler chickenSix strains were used in the present study. strain with high adhesion was selected, theacid and bile salt tolerance of the strain was further evaluated. The results showed that Lfermentum1.2029showed good adhesive ability and was able to survive in low pHconditions and in the presence of bile salts. To study the mucin expression of the smallintestine epithelium of broilers after the strain was orally administrated. Twenty four1-d-oldArbor Acres broilers were blocked into2experimental treatments: control group andlactobacillus treatment group. Fresh prepared aliquots (500μL,108CFU) of L. fermentum1.2029were administered to the treatment group birds via orogastric inoculation. Accordingly,the control group birds were inoculated with the same volume of PBS. The results showedthat the strain significantly increased goblet cell density in the jejunum and did not affectgoblet cell density of other intestinal segments. The treatment group with administration oflactobacillus induce a3.89-fold and3.32-fold increase in MUC2mRNA level in the jejunum and in the ileum compared with the controls (P <0.05), respectively. In the duodenum, mucinmRNA expression tended to increase after lactobacillus treatment, although the increase wasnonsignificant. The study suggests that L. fermentum1.2029can protect intestinal mucus bystimulating Muc2expessionTrial2. Effect of Lactobacillus fermentum1.2029on intestinal mucosal immuneresponse in Necrotic Enteritis of broiler chickensTo investigate the anti-inflammatory properties of L.fermentum1.2029in NE of chickens.Two hundred and fouty1-day-old male Arbor Acres broilers were blocked into3experimentalgroups: normal group, NE model group and L. fermentum treatment group. L. fermentum1.2029(1.0mLd-1,108CFU mL-1) was orally administered daily to L. fermentum treatmentgroup during the course of the experiment. Accordingly, all uninfected normal chickens wereinoculated with the same volume of PBS. C. perfringens (0.5mL on d1and1.0mL on d14–21,108CFU mL-1) was administered to chickens in NE model group. At28d, samples werecollected for histological scoring and mRNA analysis. Gross NE lesion scores were alsoperformed. The results showed that L. fermentum1.2029reduced NE lesion severity inchickens. Histological scores revealed that the control group revealed intact mucosa.Intestinal specimens from C. perfringens challenge group exhibited strong inflammatorydamage, whereas in L. fermentum1.2029treatment group, these symptoms were attenuated.The results from real time-PCR analysis showed that C. perfringens increased mRNAexpression of TLR2and IFN-γ by two fold and three fold, respectively. L. fermentum1.2029treatment down-regulated the level of TLR2and IFN-γ (P <0.05). The expression of IL-10was decreased after C. perfringens infection, L. fermentum1.2029treatment up-regulated thelevel of IL-10(P <0.05). TLR4had no significantly changes among three groups. The resultsshowed that L. fermentum1.2029could regulate intestinal mucosal immune response andameliorate inflammation by changing expression of cytokine and TLR expression.Trial3. The effect of L. fermentum1.2029on intestinal epithelial restitution inNecrotic Enteritis of the broiler chickensTo investigate effect of L. fermentum1.2029on intestinal epithelial restitution inNecrotic Enteritis of the Broiler Chickens, Two hundred and fouty1-day-old maleArbor Acres broilers were blocked into3experimental groups: normal group, NE modelgroup and L. fermentum treatment group. L. fermentum1.2029(1.0mLd-1,108CFU mL-1) wasorally administered daily to L. fermentum treatment group during the course of the experiment.Accordingly, all uninfected normal chickens were inoculated with the same volume of PBS. C.perfringens (0.5mL on d1and1.0mL on d14–21,108CFU mL-1) was administered tochickens in NE model group. The results showed that L. fermentum1.2029promoted the migration of enterocytes along the crypt-villus axis in NE of chickens (P <0.05). Enzymaticactivity of sucrase and the expression of cdx1were increased by L. fermentum1.2029(P <0.05). L. fermentum1.2029treatment increased significantly the expression of EGFR in ileummucosa when compared with NE model and the EGF concentrations in plasma (P <0.05), butdid not affect ability of cell proliferation in crypt. In conclusion, L. fermentum1.2029couldaccelerate intestinal epithelial restitution in case of NE. The restitution process wasaccompanied with differentiation of the epithelium.Trial4. Effect of L. fermentum1.2029on expression of tight junction protein innecrotic enteritis of chickensTo investigate effect of Lactobacillus fermentum1.2029on expresion of tight junctionprotein in necrotic enteritis (NE) of chickens,2401-d-old Arbor Acres broiler chicks wererandomly separated into3groups: normal group, NE model group and L. fermentumtreatment group. At28d, chickens were killed, and ileal segments of24birds from eachexperimental group were collected for observation by electron microscope and mRNAanalysis by real-time PCR. The results showed L. fermentum1.2029improved damage ofmicrovilli in NE, L. fermentum1.2029increased level of claudin-1and occludin in NE ofchickens (P <0.05). The results indicated that L. fermentum1.2029had protective functionfor intestinal barrier更多还原