【作者】 常桂芳；

【导师】 徐子伟； 乐国伟；

【作者基本信息】 江南大学 ， 食品营养与安全， 2009， 博士

【摘要】 派伊尔结(Peyer’s patch,PP)是肠道黏膜免疫系统的重要部位,是乳酸菌进入肠黏膜免疫诱导部位的主要通道。本课题研究乳酸菌表面性质和黏附黏液、PP性能的关系,不同黏附性能的乳酸菌和强黏附菌株(三种状态:活、紫外灭活和热灭活)的免疫调节作用,强黏附菌株的抗感染作用和调节PP基因表达的作用。这些研究有助于阐明乳酸菌调节黏膜免疫机制,也将为乳酸菌免疫调节制剂的开发提供理论依据。试验先用大鼠黏液、小鼠PP为黏附基质,研究23株细菌的黏附性,结果显示,Lactobacillus plantarum Fn008、Lactobacillus acidophilus Fn037、Lactobacillus rhamnosus GG(LGG)和Salmonella Typhimurium为强黏附菌株。利用细菌黏附正十六烷、水接触角法测定细菌表面疏水性,结果表明,23株细菌的表面疏水性与黏附性没有相关性。利用红细胞凝集试验分析乳酸菌细胞表面凝集素的表达情况,结果表明23株的细菌凝集性与其黏附性没有相关性。利用细菌表面疏水性、表面电荷和液体表面张力计算细菌表面自由能,结果表明22株乳酸菌的表面自由能与其黏附PP性呈负相关。为了研究乳酸菌黏附PP能力与免疫调节的关系,分别给小鼠连续3天灌胃四株黏附PP能力不同的乳酸菌(LGG、Fn008、Fn022和Fn036)以及Salmonella,检测脾淋巴细胞增殖能力及其基因IL-12表达能力、腹腔和派伊尔结巨噬细胞吞噬能力、细胞因子(IL-4,IL-10,IFN-γ,TGF-β,IL-17)、黏膜免疫指标sIgA、免疫相关基因(CD80,Mfge8,CXCL13和CD79a)及细菌易位,结果表明:乳酸菌(LGG、Fn008、Fn022和Fn036)黏附派伊尔结能力与其增强PP巨噬细胞吞噬活性及产生sIgA能力呈正相关;乳酸菌(LGG、Fn008、Fn022和Fn036)可诱导抗原呈递细胞特异基因(CD80和Mfge8)和B细胞趋化因子CXCL13表达上调,同时可抑制B细胞特异性基因CD79a表达,这种作用依赖于其黏附PP的能力;乳酸菌和Salmonella都诱导了Th1免疫;乳酸菌Fn008、Fn022、LGG和Fn036没有破坏肠道黏膜屏障,而Salmonella却破坏肠道黏膜屏障引起细菌显著易位。为了研究乳酸菌Fn008经过不同处理后黏附PP能力与其免疫调节作用的关系,分别给小鼠灌胃活菌Fn008 (V-Fn008)、紫外灭活菌Fn008 (UV-Fn008)、热灭活菌Fn008 (HK-Fn008)、Fn008+Salmonella和Salmonella,连续3天,检测脾淋巴细胞增殖能力及其基因IL-12表达能力、腹腔和派伊尔结巨噬细胞吞噬能力、细胞因子(IL-4,IL-10,IFN-γ,TGF-β,IL-17)、黏膜免疫指标sIgA、免疫相关基因(CD80,Mfge8,CXCL13和CD79a)及细菌易位,结果表明:V-Fn008、UV-Fn008和HK-Fn008黏附PP的能力与其增强PP巨噬细胞吞噬能力及产生sIgA的能力呈正相关。V-Fn008、UV-Fn008和HK-Fn008可诱导抗原呈递细胞特异基因(CD80和Mfge8)和B细胞趋化因子CXCL13表达,同时可抑制B细胞特异性基因CD79a表达,这种作用依赖于其黏附派伊尔结的能力。口服V-Fn008、UV-Fn008和HK-Fn008都诱导了Th1细胞免疫,且UV-Fn008和HK-Fn008诱导的Th1免疫反应弱于V-Fn008;活菌Fn008能抑制Salmonell引起的细菌易位;利用On-tools分析口服乳酸菌Fn008小鼠空肠派伊尔结显著改变的基因,研究相关的显著性通路,结果表明,口服乳酸菌Fn008引起空肠派伊尔结内的响应基因主要与免疫、黏膜屏障和其它通路(肌动蛋白骨架的调节和自然杀伤性细胞介导的细胞毒性等)通路有关。采用定量PCR结果表明,乳酸菌(LGG、Fn008、Fn022、Fn036、UV-Fn008和HK-Fn008)可诱导派伊尔结黏着斑通路中Itgb1、lamaα3和Thbs2三个基因表达上调,这种作用依赖于其黏附派伊尔结的能力;乳杆菌Fn008通过使黏着斑中的基因FAK表达下调,而上调Itgb1、lamaα3、Itgb4、Thbs2基因表达,从而抑制沙门氏菌引起的细菌易位。总之,本研究结果表明,特定乳酸菌主要通过表面自由能介导的作用力黏附PP,其黏附PP性与其增强派伊尔结巨噬细胞吞噬活性及诱导sIgA产生的能力呈正相关;乳酸菌可诱导派伊尔结黏着斑通路中Itgb1、lamaα3和Thbs2三个基因表达上调、可诱导抗原呈递细胞特异基因CD80和Mfge8表达上调,同时可抑制B细胞特异性基因CD79a表达,这种作用依赖于其黏附派伊尔结的能力;乳杆菌Fn008通过使黏着斑中的基因FAK表达下调,而上调Itgb1、Itgb4、lamaα3、Thbs2基因表达,从而抑制沙门氏菌引起的细菌易位。更多还原

【Abstract】 Peyer’s patches (PP) are the main site of intestinal mucosa immunity system through which Lactobacillus come into contact with intestinal mucosal immunity system to modulate their functions.This study was focused on relationship with adhesion of Lactobacillus to immobilized mucus from rat small intestine or mice PP in vitro and Lactobacillus surface properties, Lactobacillus with different adhesive properties or treatment (three states: live,ultraviolet (UV)-inactivated and heat-killed(HK)) in the role of immune regulation, anti-infection and regulation of gene expression in PP with strong adhesive strain.The aim was to give some explanation on mechanism that Lactobacillus modulates mucosal immunity and to help developing immunomodulatory agents related to Lactobacillus.Adhesive properties of twenty-three bacteria were studied by using immobilized mucus from rat or PP under spectroscopic methodology or radioactivity.The results indicted that Lactobacillus plantarum Fn008, Lactobacillus acidophilus Fn037, Lactobacillus rhamnose GG (LGG) and Salmonella Typhimurium were the strong adhesive strains. Bacterial surface hydrophobicity was studied by hexadecane (BATH) and water contact angle method. Bacterial surface electric charge was determined by Zeta potential. Surface free energy was calculated by surface tension, electric charge and water contact angle. Expression of lectin was studied by hemagglutination of rabbit red cells.The results indicated that surface free energy of Lactobacillus had a negative correlation with adhesion ability to PP. Expression of lectin on bacterial surface was studied by hemagglutination of rabbit red cells.The results indicated that there was no correlation with hemagglutination of Lactobacillus and adhesion ability.In order to determine the relationship between the immune modulation and adhesion ability, LGG, Fn008, Fn022 and Fn036 were oral administrated to mice with consecutive 3 days.Spleen lymphocyte proliferation and IL-12 gene expression, phagocytosis of peritoneal and PP macrophage, cytokine (IL-4, IL-10, IFN-γ, TGF-β, IL-17), mucosal immunity indicators sIgA, immune-related genes (CD80,Mfge8,CXCL13 and CD79a)and bacterial translocation were analysised respectively.The results indicated that adhesion ability with PP of Lactobacillus(LGG,Fn008,Fn022 and Fn036) was correlated with macrophage phagocytic activity in PP and ability to production of sIgA. Lactobacillus (LGG, Fn008, Fn022 and Fn036) could induce antigen presenting cell specific genes(CD80 and Mfge8)and B-cell chemokines (CXCL13)expression up-regulated,but inhibit B cell specific gene CD79a expression.This function was dependent on adhesion ability to PP.The immune response induced by Lactobacillus was strong than Salmonella; they all induced Th1 immune response. Lactobacillus (Fn008, Fn022, LGG and Fn036) could not destroy mucosa barrier, but Salmonella could cause bacterial translocation significantly through destroying mucosa barrier.In order to determine the relationship between the immune modulation and adhesion ability of different-treated Fn008, V-Fn008,UV-Fn008, HK-Fn008,Fn008+Salmonella and Salmonella were oral administrated to mice with consecutive 3 days. Spleen lymphocyte proliferation and IL-12 gene expression, phagocytosis of peritoneal and PP macrophage, cytokine (IL-4, IL-10, IFN-γ, TGF-β, IL-17), mucosal immunity indicator sIgA, immune-related genes (CD80, Mfge8, CXCL13 and CD79a) and bacterial translocation were analysised respectively.The results indicated that adhesion ability with PP of V-Fn008, UV-Fn008 and HK-Fn008 were correlated with macrophage phagocytic activity in PP and ability to production of sIgA.V-Fn008,UV-Fn008 and HK-Fn008 could induce antigen presenting cell specific genes(CD80 and Mfge8)and B-cell chemokines (CXCL13)expression up-regulated,but inhibit B cell specific gene CD79a expression.This function was dependent on the adhesion ability to PP.The immune response induced byUV-Fn008 and HK-Fn008 was weaker than V-Fn008; they all induced Th1 immune response. Lactobacillus (Fn008, Fn022, LGG and Fn036) could not destroy mucosa barrier, but Salmonella could cause bacterial translocation significantly through destroying mucosa barrier. V-Fn008 can inhibit bacterial translocation induced by Salmonella.Onto-Tools were applied to explore significant genes in jejunal Peyer’s patch induced by oral administration Fn008 with consecutive 14 days. The results indicated that responsive genes in jejunal Peyer’s patch mainly related to immunity, mucosal barrier and other pathways (regulation of actin cytoskeleton, natural killer cell-mediated cytotoxicity and etc.). The results of quantitative PCR indicated that Lactobacillus (LGG,Fn008,Fn022,Fn036, UV-Fn008 and HK-Fn008) could induce focal adhesion gene(Itgb1,lamaα3 and Thbs2) expression upregulated.This function was dependent on the adhesion ability to PP.V-Fn008 can induce key gene FAK down-regulated and genes ( Itgb1,lamaα3,Itgb4,Thbs2) up-regulated in focal adhesion.This caused inhibition of bacterial translocation induced by Salmonella.In conclusion, the results of present study had revealed that adhesive ability of Lactobacillus to PP mainly determined by surface free energy.Its adhesive ability to PP had correlation with macrophage phagocytic activity in PP and ability to production sIgA. Lactobacillus can induce focal adhesion gene(Itgb1,lamaα3 and Thbs2),antigen presenting cell specific genes(CD80 and Mfge8)and B-cell chemokines (CXCL13)expression up-regulated,but inhibit B cell specific gene CD79a expression.This function was dependent on the adhesion ability to PP. V-Fn008 can induce key gene FAK down-regulated and genes (Itgb1,lamaα3,Itgb4,Thbs2) up-regulated in focal adhesion.This caused inhibition of bacterial translocation induced by Salmonella.更多还原