由Toll like receptors介导的布拉氏酵母菌和枯草芽孢秆菌B10对肉鸡肠黏膜免疫功能影响的研究

摘要

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，20 mg/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;显著提高肉鸡血清中谷胱甘肽过氧化物酶、谷胱甘肽还原酶、过氧化物酶、过氧化氢酶活性及谷胱甘肽、白蛋白和低密度脂蛋白含量，使丙二醛、尿酸和甘油三酯含量显著降低。布拉氏酵母和枯草芽孢杆菌B10对肉鸡肠道结构和微生物菌群结构的影响。益生菌组肉鸡法氏囊和胸腺相对重量、肠绒毛高度、杯状细胞数量均显著高于对照组。添加益生菌显著提高了肠道紧密连接蛋白occluding、cloudin2及cloudin3mRNA表达水平。肠道菌群分析表明，益生菌组空肠和回肠内菌群数量显著增加，厚壁菌门的数量处于优势地位，而肠道内菌群多样性显著降低。
　　 布拉氏酵母和枯草芽孢杆菌B10对肉鸡肠道免疫功能及TLR信号通路基因mRNA表达的影响。与对照组相比，酵母菌处理组空肠和回肠黏膜中IL-1β TNFα、IL-7、IL-6、IL-4、IL-10和TGF-β和芽孢杆菌组空肠和回肠黏膜TNFα、IL-17、IL-6和TGF-β水平显著提高。益生菌处理组空肠黏膜INF-γ、IL-8含量及回肠黏膜INF-γ含量均显著低于对照组。此外，益生菌处理组空肠IgA阳性细胞，血清IgA、sIgA水平显著提高，其中酵母菌处理组IgG含量显著高于对照组。益生菌处理组肉鸡空肠黏膜Toll-样受体TLR2、TLR4、TLR15和相关信号蛋白MyD88、TRAF6、TAB2和NF-κB基因表达水平显著上调。
　　 布拉氏酵母菌和枯草芽孢杆菌B10对鸡骨髓源树突状细胞(DCs)免疫功能的影响。树突状细胞(DCs)是家禽调节免疫功能的重要靶细胞，但目前体外培养技术仍不成熟。本研究首先利用E.coli BL21成功表达了鸡粒细胞-巨噬细胞集落刺激因子(chi-rGMCSF)，并建立了培养肉鸡DC细胞的方法。随后研究了布拉氏酵母菌和枯草芽孢杆菌B10对鸡骨髓源树突状细胞(DCs)免疫功能的影响。DCs细胞分别与PBS(Ctr组)、LPS（LPS组）、布拉氏酵母菌（Sb组）、芽孢杆菌组(Bs组)、布拉氏酵母菌+芽孢杆菌组(Sb+Bs组)共孵育3h、6h、12 h后进行分析。扫描电镜显示益生菌能够粘附于DCs表面，透射电子显微镜进一步观察发现DCs能够有效吞噬和降解益生菌。与对照组相比，各处理组细胞表面分子MHC-Ⅱ、CD40、CD80和CD86基因表达水平均显著上调。此外， TLR1、TLR2、TLR4、TLR15和相关信号蛋白MyD88、TRAF6、TAB1和NFκ-B基因表达水平也显著提高。与对照组相比，Sb组、Bs组和Sb+Bs组中IL-1β、IL-17、IL-4、TGF-β和IL-10含量显著增加而INF-γ、IL-8的含量则显著降低。LPS处理组IL-12、INF-γ、IL-8含量显著高于对照组。
　　 综上所述，布拉氏酵母菌和枯草芽孢杆菌B10能够通过TLR介导的信号通路有效调节肉鸡肠道和鸡骨髓源树突状细胞免疫功能，并通过改善肠道结构和菌群组成促进肉鸡生长。

目录

声明

Dedication

ACKNOWLEDGEMENTS

论文说明

CONTENTS

LIST OF TABLES

LIST OF FIGURES

ABSTRACT

摘要

ABBREVIATIONS

CHAPTER-Ⅰ INTRODUCTION

CHAPTER-ⅡREVIEW OF LITERATURE

2．1 Beneficial effects of probiotics 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

2．2 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 on growth performance，digestive enzyme activities，antioxidation capacity and blood homeostasis 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 functioning

3．3．5 Blood biochemical examination

3．4 DISCUSSION

3．5 CONCLUSION

CHAPTER-Ⅳ Effect of Saccharomyces boulardii and Bacillus B10 on intestinal ultrastructure 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 synthesis

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 cytokines by ELISA

4．2．10 DNA isolation from fecal contents

4．2．11 PCR and pyrosequencing

4．2．12 Post-run analysis

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-Ⅳ The effect of Saccharomyces boulardii and Bacillus subtilis B10 on intestinal immunity 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

5．6 CONCLUSION

CHAPTER-Ⅵ Cloning approach coordinated with IL-4 to asses optimal growth and biological activity 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 chicken bone 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 Extranction 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 surface 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