由Toll like receptors介導(dǎo)的布拉氏酵母菌和枯草芽孢桿菌B10對肉雞腸黏膜免疫功能影響的研究
發(fā)布時間：2021-08-30 14:29
研制安全、高效、綠色的飼用添加劑產(chǎn)品以替代抗生素,是保障畜牧業(yè)健康發(fā)展的必然趨勢。益生菌制劑因其具有效果顯著、安全性好等諸多優(yōu)點已成為替代飼用抗生素的首選。布拉氏酵母和枯草芽孢桿菌是應(yīng)用較為廣泛的益生菌,能改善腸道健康、促進動物生長以及提高宿主的免疫力。盡管益生菌的免疫調(diào)節(jié)作用越來越受關(guān)注,但是到目前為止,機制尚未清楚。本論文以布拉氏酵母和枯草芽孢桿菌B10為材料,通過體外試驗和體內(nèi)試驗研究其對肉雞腸黏膜先天免疫的調(diào)節(jié)作用及可能機理。主要的研究內(nèi)容和結(jié)果如下：布拉氏酵母和枯草芽孢桿菌B10對肉雞生長、消化酶活性、抗氧化功能和血清生化指標(biāo)的影響。試驗選用1日齡三黃肉雞300羽,隨機分為三組,每組設(shè)5個重復(fù),每個重復(fù)20羽。對照組飼喂含抗生素（維吉尼亞霉素,20mg/kg）的基礎(chǔ)日糧,處理組在每千克基礎(chǔ)日糧（不添加抗生素）中分別添加1×108CFU/克飼料的布拉氏酵母（Bs）和枯草芽孢桿菌（Sb）。飼養(yǎng)試驗為期72天。結(jié)果表明,與對照組相比,飼糧中添加益生菌能顯著促進肉雞生長,提高肉雞空腸Na+/K+ATP酶、脂肪酶和γ-谷氨酰轉(zhuǎn)肽酶活性,Sb還可以提高回腸中γ-谷氨酰轉(zhuǎn)肽酶活性；顯著提高...
【文章來源】：浙江大學(xué)浙江省 211工程院校 985工程院校 教育部直屬院校
【文章頁數(shù)】：183 頁
【學(xué)位級別】：博士
【文章目錄】：
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
【參考文獻(xiàn)】：
期刊論文
[1]枯草芽孢桿菌對肉雞生長性能和腸黏膜抗氧化及免疫功能的影響[J]. 李衛(wèi)芬,文靜,吳紅照,翟玲,余東游. 中國畜牧雜志. 2011(09)
[2]屎腸球菌對仔豬生長性能、免疫和抗氧化功能的影響[J]. 文靜,孫建安,周緒霞,李衛(wèi)芬. 浙江農(nóng)業(yè)學(xué)報. 2011(01)
[3]枯草芽孢桿菌對肉雞生長性能及其抗氧化和免疫功能的影響[J]. 余東游,毛翔飛,秦艷,李衛(wèi)芬. 中國畜牧雜志. 2010(03)
[4]地衣芽孢桿菌對三角帆蚌消化酶活性、免疫指標(biāo)和抗氧化指標(biāo)的影響[J]. 沈文英,余東游,李衛(wèi)芬,羅文,祝堯榮. 動物營養(yǎng)學(xué)報. 2009(01)
本文編號：3372952
【文章來源】：浙江大學(xué)浙江省 211工程院校 985工程院校 教育部直屬院校
【文章頁數(shù)】：183 頁
【學(xué)位級別】：博士
【文章目錄】：
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
【參考文獻(xiàn)】：
期刊論文
[1]枯草芽孢桿菌對肉雞生長性能和腸黏膜抗氧化及免疫功能的影響[J]. 李衛(wèi)芬,文靜,吳紅照,翟玲,余東游. 中國畜牧雜志. 2011(09)
[2]屎腸球菌對仔豬生長性能、免疫和抗氧化功能的影響[J]. 文靜,孫建安,周緒霞,李衛(wèi)芬. 浙江農(nóng)業(yè)學(xué)報. 2011(01)
[3]枯草芽孢桿菌對肉雞生長性能及其抗氧化和免疫功能的影響[J]. 余東游,毛翔飛,秦艷,李衛(wèi)芬. 中國畜牧雜志. 2010(03)
[4]地衣芽孢桿菌對三角帆蚌消化酶活性、免疫指標(biāo)和抗氧化指標(biāo)的影響[J]. 沈文英,余東游,李衛(wèi)芬,羅文,祝堯榮. 動物營養(yǎng)學(xué)報. 2009(01)
本文編號：3372952
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