鼠李糖乳杆菌肽聚糖对鸡组织细胞β-防御素9基因表达的影响

发布时间：2018-03-07 08:49

本文选题：鼠李糖乳杆菌MLGA　切入点：完整肽聚糖　出处：《江西农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：防御素是组成生物体天然免疫的重要成分之一,防御素不仅具有广谱抗菌作用,还可作为细胞间免疫效应分子通过多重机制发挥免疫调节功能和抗感染作用,然而有关防御素发挥抗感染作用和防御素表达的调控机制仍未完全阐明。本试验从鼠李糖乳杆菌MLGA细胞壁分离提取完整肽聚糖(whole peptidoglycan,WPG),研究不同剂量WPG对鸡组织(细胞)中禽β-防御素9(avianβ-defensin 9,AvBD9)基因表达的影响,并比较病原菌来源肽聚糖与益生菌来源肽聚糖对AvBD9表达调节作用的差异。同时,测定鼠李糖乳杆菌WPG对免疫细胞炎症细胞因子的表达以确定WPG诱导细胞AvBD9表达是否为炎症细胞因子非依赖性。最后,测定免疫细胞与鼠李糖乳杆菌WPG共培养后细胞裂解液的抗菌作用。试验结果如下:(1)采用三氯乙酸(trichloroacetic acid,TCA)法分离提取鼠李糖乳杆菌MLGA细胞壁WPG样品,WPG的提取率为3.89%。经电镜观察、氨基酸组成成分分析和溶菌酶消化试验鉴定所提取的物质为鼠李糖乳杆菌MLGA细胞壁完整肽聚糖。(2)鼠李糖乳杆菌MLGA细胞壁WPG可不同程度地促进鸡外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)、脾细胞、胸腺细胞、肝细胞及鸡胚盲肠、回肠和空肠AvBD9 mRNA的表达,且呈剂量依赖性。不同浓度WPG对AvBD9mRNA表达的促进作用在不同细胞之间、不同肠段之间存在差异。与对照组相比,病原菌金黄色葡萄球菌肽聚糖降低了鸡PBMCs和脾细胞AvBD9 mRNA的表达,鼠李糖乳杆菌MLGA细胞壁WPG及其酶解产物上调了鸡PBMCs和脾细胞AvBD9mRNA的表达。鼠李糖乳杆菌MLGA细胞壁WPG对鸡PBMCs和脾细胞炎症细胞因子IL-1β、IL-8和IL-12p40的表达无显著影响(P0.05),而WPG酶解产物不同程度地降低炎症细胞因子IL-1β、IL-8和IL-12p40的表达(P0.05或P0.01)。肽聚糖及其酶解产物对AvBD9基因表达的促进作用是炎症细胞因子非依赖性的。(3)不同浓度WPG处理PBMCs或脾细胞后其细胞裂解液均极显著抑制肠炎沙门氏菌的生长(P0.01),且细胞裂解液的抑菌作用随着WPG浓度的升高而提高,呈剂量依赖性。WPG本身及WPG与PBMCs和脾细胞共培养所得的细胞培养上清液对肠炎沙门氏菌生长无抑制作用。综上所述,鼠李糖乳杆菌完整肽聚糖可促进鸡免疫细胞和肠组织AvBD9基因表达,且呈时间依赖性和剂量依赖性。益生菌鼠李糖乳杆菌肽聚糖及其酶解产物提高了细胞AvBD9基因的表达,且对AvBD9基因表达的促进作用是炎症细胞因子非依赖性的。相反,病原菌金黄色葡萄球菌肽聚糖降低鸡免疫细胞AvBD9基因的表达。鼠李糖乳杆菌肽聚糖及其酶解产物可通过上调鸡组织细胞尤其是免疫细胞β-防御素如AvBD9表达而发挥益生作用,提高鸡抗感染天然免疫功能。
[Abstract]:Defensin is one of the important components of innate immune, defensins have broad-spectrum antibacterial effect not only, also can be used as cell immune effector molecules through multiple mechanisms play a role in regulating immune function and anti infection effect, but the defensins play the role of anti infection and defense has not yet fully elucidated. The expression of the separation test whole peptidoglycan from Lactobacillus rhamnosus MLGA cell wall (whole peptidoglycan, WPG). The effects of different doses of WPG in chicken tissues (cells) in avian beta defensin 9 (avian -defensin 9 beta, AvBD9) gene expression, and compare the source pathogen peptidoglycan and probiotics source peptidoglycan expression the role of AvBD9. At the same time, the expression of Lactobacillus rhamnosus WPG on immune inflammatory cytokines were measured to determine whether AvBD9 expression induced by WPG cells for inflammatory cytokine dependent. After the determination of the antibacterial effect of immune cells and rhamnose lactobacillus WPG after co cultured cell lysate. The results are as follows: (1) using three chloroacetic acid (trichloroacetic acid, TCA) was isolated from Lactobacillus rhamnosus MLGA cell wall WPG samples, the extraction rate of WPG 3.89%. by electron microscope, amino acid composition analysis and lysozyme digestion test identified the extracted material for Lactobacillus rhamnosus MLGA of whole peptidoglycan. (2) of Lactobacillus rhamnosus MLGA cell wall WPG can significantly promote chicken peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs), spleen cells, thymus, liver chick embryo cells and expression of cecum, jejunum and ileum in AvBD9 mRNA, in a dose-dependent manner. Different concentrations of WPG on the expression of AvBD9mRNA in different role between cells between different segments. The difference compared with the control group, the pathogen of golden Aureus peptidoglycan decreased the expression of chicken PBMCs and spleen cells of AvBD9 mRNA, Lactobacillus rhamnosus MLGA cell wall WPG and its hydrolysates up-regulated the expression of chicken PBMCs and spleen cells of AvBD9mRNA. Lactobacillus rhamnosus MLGA cell wall WPG of chicken spleen cells PBMCs and inflammatory cytokines of IL-1 beta expression had no significant effects IL-8 and IL-12p40 (P0.05), and WPG of hydrolysate decreased in different degree of inflammatory cytokines IL-1 beta, expression of IL-8 and IL-12p40 (P0.05 or P0.01). The peptidoglycan and its hydrolysates on the expression of AvBD9 gene promotes inflammatory cytokine dependent. (3) WPG with different concentration of PBMCs after the spleen cells or cell lysates were significantly inhibited the growth of Salmonella enteritidis (P0.01), and the inhibition of cell lysate with the increase of concentration of WPG increased in a dose dependent manner,.WPG itself and WPG and PBMCs and spleen cell co culture Keep the cell culture supernatant on Salmonella enteritidis growth inhibitory effect. In summary, Lactobacillus rhamnosus whole peptidoglycan can promote the expression of AvBD9 gene of chicken immune cells and intestinal tissue, and in a time and dose dependent. Probiotic Lactobacillus rhamnosus poly peptide and sugar enzyme to improve the expression of AvBD9 gene hydrolysates, and the expression of AvBD9 gene promotes inflammatory cytokine dependent. On the contrary, the pathogenic bacteria Staphylococcus aureus peptidoglycan reduced expression of chicken immune cells AvBD9 gene. The product of Lactobacillus rhamnosus peptidoglycan and its enzyme can upregulate the chicken tissues especially the immune cells of beta defensins such as the expression of AvBD9 and probiotic, improve chicken anti infection immune function.

【学位授予单位】：江西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

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