由Toll like receptors介导的布拉氏酵母菌和枯草芽孢杆菌B10对肉鸡肠黏膜免疫功能影响的研究
发布时间：2021-08-30 14:29
研制安全、高效、绿色的饲用添加剂产品以替代抗生素,是保障畜牧业健康发展的必然趋势。益生菌制剂因其具有效果显著、安全性好等诸多优点已成为替代饲用抗生素的首选。布拉氏酵母和枯草芽孢杆菌是应用较为广泛的益生菌,能改善肠道健康、促进动物生长以及提高宿主的免疫力。尽管益生菌的免疫调节作用越来越受关注,但是到目前为止,机制尚未清楚。本论文以布拉氏酵母和枯草芽孢杆菌B10为材料,通过体外试验和体内试验研究其对肉鸡肠黏膜先天免疫的调节作用及可能机理。主要的研究内容和结果如下：布拉氏酵母和枯草芽孢杆菌B10对肉鸡生长、消化酶活性、抗氧化功能和血清生化指标的影响。试验选用1日龄三黄肉鸡300羽,随机分为三组,每组设5个重复,每个重复20羽。对照组饲喂含抗生素（维吉尼亚霉素,20mg/kg）的基础日粮,处理组在每千克基础日粮（不添加抗生素）中分别添加1×108CFU/克饲料的布拉氏酵母（Bs）和枯草芽孢杆菌（Sb）。饲养试验为期72天。结果表明,与对照组相比,饲粮中添加益生菌能显著促进肉鸡生长,提高肉鸡空肠Na+/K+ATP酶、脂肪酶和γ-谷氨酰转肽酶活性,Sb还可以提高回肠中γ-谷氨酰转肽酶活性；显著提高...
【文章来源】：浙江大学浙江省 211工程院校 985工程院校 教育部直属院校
【文章页数】：183 页
【学位级别】：博士
【文章目录】：
ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES
ABSTRACT
摘要
ABBREVIATIONS
CHAPTER-Ⅰ INTRODUCTION
CHAPTER-Ⅱ REVIEW OF LITERATURE
2.1 Beneficial effects ofprobiotics on intestinal health
2.1.1 Helpful in lactose intolerance
2.1.2 Prevention of diarrhea
2.1.3 Reprieve from constipation
2.1.4 Colon cancer
2.1.5 Inflammatory bowel disease
2.1.6 Necrotizing enterocolitis
2.1.7 Beneficial effects for homeostasis
2.2 Effect of probiotics on intestinal immunity and chicken health
2.3 Mechanisms of Probiotic Action
2.4 Mechanism of action in intestinal defense
2.4.1 Antagonistic effects against pathogens
2.4.2 Competitive exclusion along the epithelium
2.4.3 Improving epithelial structure
2.5 Boost intestinal immunity
2.6 Importance of intestinal cells receptor signaling
2.7 Cytokine production in response of TLRs signaling
2.8 In vitro culture of chicken bone marrow derived dendritic cells
2.8.1 Dendritic cells and their sub types
2.9 Biological function of DCs
2.10 Effect of probiotics on DCs functionality
CHAPTER-Ⅲ Supplementary effects of Saccharomyces boulardii and Bacillus subtilis B10 ongrowth performance, digestive enzyme activities, antioxidation capacity and bloodhomeostasis in broilers
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.2.1 Culturing of probiotics
3.2.2 Experiment design and feeding method
3.2.3 growth perfermance and internal organs weight
3.2.4 Blood collection
3.2.5 Sampling of digesta and liver
3.2.6 Enzymatic activities analysis
3.2.7 Antioxidation capacity assays
3.2.8 Statistical analysis
3.3 RESULTS
3.3.1 Growth performance
3.3.2 Digestive enzymes activity assay
3.3.3 Serum antioxidation analysis
3.3.4 Liver antioxidation firnctioning
3.3.5 Blood biochemical examination
3.4 DISCUSSION
CHAPTER-Ⅳ Effect of Saccharomyces boulardii and Bacillus subtilis B10 on intestinalultrastructure and microbial community composition
4.1 INTRODUCTION
4.2 MATERIAL AND METHODS
4.2.1 Bacterial and yeast culture preparation
4.2.2 Experiment design and feeding procedure
4.2.3 Sampling procedure
4.2.4 Histological examination
4.2.5 Transmission electron microscopy
4.2.6 RNA extraction and cDNA svnthesis
4.2.7 Quantitative real time polymerase chain reaction (qRT-PCR)
4.2.8 Immunohistochemical staining for IgA positive cells
4.2.9 Determination of cvtokines by ELISA
4.2.10 DNA isolation from fecal contents
4.2.11 PCR and pyrosequencing
4.2.12 Post-run analvsis
4.2.13 Statistical Analyses
4.3 RESULTS
4.3.1 Histological observation
4.3.2 Intestinal ultrastructure examination
4.3.3 Ocln, Cldn2 and Cldn3 mRNA expressions
4.3.4 Microbial community diversity and species richness in the jejunum and ileum
4.3.5 Microbial coMmunity structure modulation in the Jejunum
4.3.6 Microbial community structure modulation in the Ileum
4.4 DISCUSSION
4.5 CONCLUSION
CHAPTER-Ⅴ The effect of Saccharomyces boulardii and Bacillus subtilis B10 on intestinalimmunity and mRNA expression of TLR signaling pathway in broiler
5.1 INTRODUCTION
5.2 MATERIALS AND METHODS
5.2.1 Preparation of probiotics
5.2.2 Experimental Designing
5.2.3 Sampling procedure
5.2.4 Determination of cytokines by ELISA
5.2.5 Immunohistochemistry of jejunum and ileum
5.2.6 RNA extraction and cDNA synthesis
5.2.7 Quantitative real time polymerase chain reaction (qRT-PCR)
5.2.8 Statistical analyses
5.3 RESULTS
5.3.1 Cytokine production level
5.3.2 IgA positive cells and sIgA concentration in the jejunum and ileum
5.3.3 Serum Immunoglobulin Assay
5.3.4 Surface Toll like receptor and down streaming expressions response
5.4 DISCUSSION
CHAPTER-Ⅵ Cloning approach coordinated with IL-4 to asses optimal growth and biologicalactivity of bone marrow derive denderitic cells
6.1 INTRODUCTION
6.2 MATERIALS AND METHODS
6.2.1 Cloning of chicken rGM-CSF and construction of expression vector
6.2.2 Chicken rGM-CSF protein expression and purification
6.2.3 SDS-PAGE and western blotting
6.2.4 Isolation of progenitor cells from chicken bone marrow
6.2.5 Growth and maturation of dendrite cells (DCs)
6.2.6 Cell yield evaluation
6.2.7 Morphological examination
6.2.8 Extraction of RNA and cDNA Synthesis
6.2.9 Quantitative real time polymerase chain reaction (qRT-PCR)
6.2.10 Bone marrow cells, developmental and morphological changes
6.2.11 Statistical analysis
6.3 RESULTS
6.3.1 Cloning, expression and purification of chicken GM-CSF
6.3.2 Cell culture yield
6.3.3 Morphological examination of cultured BMDCs
6.3.4 Phenotype of immature and mature chi-BMDCs
6.3.5 Genes expression level of surface markers
6.3.6 Inflammatory and anti-inflammatory cytokines genes expression
6.4 DISCUSSION
6.5 CONCLUSION
CHAPTER-Ⅶ Effects of Saccharomyces boulardii and Bacillus subtilis B10 on immunity of chickenbone marrow dendritic cells modulated by TLRs signaling pathway
7.1 INTRODUCTION
7.2 MATERIALS AND METHODS
7.2.1 Isolation and culturing of chicken bone marrow, dendrite cells (chi-BMDCs)
7.2.2 Probiotics and culture conditions
7.2.3 Experimental design
7.2.4 Morphological observation
7.2.5 Scan electron microscopy (SEM)
7.2.6 Transmission electron Microscopy
7.2.7 Extraction of RNA and cDNA Synthesis
7.2.8 Quantitation of mRNA qRT-PCR
7.2.9 Cytokines production by ELISA essay
7.2.10 Statistical Analysis
7.3 RESULTS
7.3.1 Morphological changes in chicken bone marrow derived dendrite cells
7.3.2 Scan electron microscopy
7.3.3 Transmission electron microscopy (TEM)
7.3.4 Gene expression of sanface markers
7.3.5 TLRs and associated factors expression response
7.3.6 Cytokines and chemokine determination
7.4 DISCUSSION
7.5 CONCLUSION
CHAPTER-Ⅷ SUMMARY, CONCLUSIONS AND SUGGESTIONS
Summary
Conclusions
Suggestions
REFRENCES
【参考文献】：
期刊论文
[1]枯草芽孢杆菌对肉鸡生长性能和肠黏膜抗氧化及免疫功能的影响[J]. 李卫芬,文静,吴红照,翟玲,余东游. 中国畜牧杂志. 2011(09)
[2]屎肠球菌对仔猪生长性能、免疫和抗氧化功能的影响[J]. 文静,孙建安,周绪霞,李卫芬. 浙江农业学报. 2011(01)
[3]枯草芽孢杆菌对肉鸡生长性能及其抗氧化和免疫功能的影响[J]. 余东游,毛翔飞,秦艳,李卫芬. 中国畜牧杂志. 2010(03)
[4]地衣芽孢杆菌对三角帆蚌消化酶活性、免疫指标和抗氧化指标的影响[J]. 沈文英,余东游,李卫芬,罗文,祝尧荣. 动物营养学报. 2009(01)
本文编号：3372952
【文章来源】：浙江大学浙江省 211工程院校 985工程院校 教育部直属院校
【文章页数】：183 页
【学位级别】：博士
【文章目录】：
ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES
ABSTRACT
摘要
ABBREVIATIONS
CHAPTER-Ⅰ INTRODUCTION
CHAPTER-Ⅱ REVIEW OF LITERATURE
2.1 Beneficial effects ofprobiotics on intestinal health
2.1.1 Helpful in lactose intolerance
2.1.2 Prevention of diarrhea
2.1.3 Reprieve from constipation
2.1.4 Colon cancer
2.1.5 Inflammatory bowel disease
2.1.6 Necrotizing enterocolitis
2.1.7 Beneficial effects for homeostasis
2.2 Effect of probiotics on intestinal immunity and chicken health
2.3 Mechanisms of Probiotic Action
2.4 Mechanism of action in intestinal defense
2.4.1 Antagonistic effects against pathogens
2.4.2 Competitive exclusion along the epithelium
2.4.3 Improving epithelial structure
2.5 Boost intestinal immunity
2.6 Importance of intestinal cells receptor signaling
2.7 Cytokine production in response of TLRs signaling
2.8 In vitro culture of chicken bone marrow derived dendritic cells
2.8.1 Dendritic cells and their sub types
2.9 Biological function of DCs
2.10 Effect of probiotics on DCs functionality
CHAPTER-Ⅲ Supplementary effects of Saccharomyces boulardii and Bacillus subtilis B10 ongrowth performance, digestive enzyme activities, antioxidation capacity and bloodhomeostasis in broilers
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS
3.2.1 Culturing of probiotics
3.2.2 Experiment design and feeding method
3.2.3 growth perfermance and internal organs weight
3.2.4 Blood collection
3.2.5 Sampling of digesta and liver
3.2.6 Enzymatic activities analysis
3.2.7 Antioxidation capacity assays
3.2.8 Statistical analysis
3.3 RESULTS
3.3.1 Growth performance
3.3.2 Digestive enzymes activity assay
3.3.3 Serum antioxidation analysis
3.3.4 Liver antioxidation firnctioning
3.3.5 Blood biochemical examination
3.4 DISCUSSION
CHAPTER-Ⅳ Effect of Saccharomyces boulardii and Bacillus subtilis B10 on intestinalultrastructure and microbial community composition
4.1 INTRODUCTION
4.2 MATERIAL AND METHODS
4.2.1 Bacterial and yeast culture preparation
4.2.2 Experiment design and feeding procedure
4.2.3 Sampling procedure
4.2.4 Histological examination
4.2.5 Transmission electron microscopy
4.2.6 RNA extraction and cDNA svnthesis
4.2.7 Quantitative real time polymerase chain reaction (qRT-PCR)
4.2.8 Immunohistochemical staining for IgA positive cells
4.2.9 Determination of cvtokines by ELISA
4.2.10 DNA isolation from fecal contents
4.2.11 PCR and pyrosequencing
4.2.12 Post-run analvsis
4.2.13 Statistical Analyses
4.3 RESULTS
4.3.1 Histological observation
4.3.2 Intestinal ultrastructure examination
4.3.3 Ocln, Cldn2 and Cldn3 mRNA expressions
4.3.4 Microbial community diversity and species richness in the jejunum and ileum
4.3.5 Microbial coMmunity structure modulation in the Jejunum
4.3.6 Microbial community structure modulation in the Ileum
4.4 DISCUSSION
4.5 CONCLUSION
CHAPTER-Ⅴ The effect of Saccharomyces boulardii and Bacillus subtilis B10 on intestinalimmunity and mRNA expression of TLR signaling pathway in broiler
5.1 INTRODUCTION
5.2 MATERIALS AND METHODS
5.2.1 Preparation of probiotics
5.2.2 Experimental Designing
5.2.3 Sampling procedure
5.2.4 Determination of cytokines by ELISA
5.2.5 Immunohistochemistry of jejunum and ileum
5.2.6 RNA extraction and cDNA synthesis
5.2.7 Quantitative real time polymerase chain reaction (qRT-PCR)
5.2.8 Statistical analyses
5.3 RESULTS
5.3.1 Cytokine production level
5.3.2 IgA positive cells and sIgA concentration in the jejunum and ileum
5.3.3 Serum Immunoglobulin Assay
5.3.4 Surface Toll like receptor and down streaming expressions response
5.4 DISCUSSION
CHAPTER-Ⅵ Cloning approach coordinated with IL-4 to asses optimal growth and biologicalactivity of bone marrow derive denderitic cells
6.1 INTRODUCTION
6.2 MATERIALS AND METHODS
6.2.1 Cloning of chicken rGM-CSF and construction of expression vector
6.2.2 Chicken rGM-CSF protein expression and purification
6.2.3 SDS-PAGE and western blotting
6.2.4 Isolation of progenitor cells from chicken bone marrow
6.2.5 Growth and maturation of dendrite cells (DCs)
6.2.6 Cell yield evaluation
6.2.7 Morphological examination
6.2.8 Extraction of RNA and cDNA Synthesis
6.2.9 Quantitative real time polymerase chain reaction (qRT-PCR)
6.2.10 Bone marrow cells, developmental and morphological changes
6.2.11 Statistical analysis
6.3 RESULTS
6.3.1 Cloning, expression and purification of chicken GM-CSF
6.3.2 Cell culture yield
6.3.3 Morphological examination of cultured BMDCs
6.3.4 Phenotype of immature and mature chi-BMDCs
6.3.5 Genes expression level of surface markers
6.3.6 Inflammatory and anti-inflammatory cytokines genes expression
6.4 DISCUSSION
6.5 CONCLUSION
CHAPTER-Ⅶ Effects of Saccharomyces boulardii and Bacillus subtilis B10 on immunity of chickenbone marrow dendritic cells modulated by TLRs signaling pathway
7.1 INTRODUCTION
7.2 MATERIALS AND METHODS
7.2.1 Isolation and culturing of chicken bone marrow, dendrite cells (chi-BMDCs)
7.2.2 Probiotics and culture conditions
7.2.3 Experimental design
7.2.4 Morphological observation
7.2.5 Scan electron microscopy (SEM)
7.2.6 Transmission electron Microscopy
7.2.7 Extraction of RNA and cDNA Synthesis
7.2.8 Quantitation of mRNA qRT-PCR
7.2.9 Cytokines production by ELISA essay
7.2.10 Statistical Analysis
7.3 RESULTS
7.3.1 Morphological changes in chicken bone marrow derived dendrite cells
7.3.2 Scan electron microscopy
7.3.3 Transmission electron microscopy (TEM)
7.3.4 Gene expression of sanface markers
7.3.5 TLRs and associated factors expression response
7.3.6 Cytokines and chemokine determination
7.4 DISCUSSION
7.5 CONCLUSION
CHAPTER-Ⅷ SUMMARY, CONCLUSIONS AND SUGGESTIONS
Summary
Conclusions
Suggestions
REFRENCES
【参考文献】：
期刊论文
[1]枯草芽孢杆菌对肉鸡生长性能和肠黏膜抗氧化及免疫功能的影响[J]. 李卫芬,文静,吴红照,翟玲,余东游. 中国畜牧杂志. 2011(09)
[2]屎肠球菌对仔猪生长性能、免疫和抗氧化功能的影响[J]. 文静,孙建安,周绪霞,李卫芬. 浙江农业学报. 2011(01)
[3]枯草芽孢杆菌对肉鸡生长性能及其抗氧化和免疫功能的影响[J]. 余东游,毛翔飞,秦艳,李卫芬. 中国畜牧杂志. 2010(03)
[4]地衣芽孢杆菌对三角帆蚌消化酶活性、免疫指标和抗氧化指标的影响[J]. 沈文英,余东游,李卫芬,罗文,祝尧荣. 动物营养学报. 2009(01)
本文编号：3372952
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/3372952.html
