双歧杆菌的完整肽聚糖激活巨噬细胞的信号机制

发布时间：2018-03-01 18:06

本文关键词： 双歧杆菌完整肽聚糖巨噬细胞蛋白激酶C 细胞外信号调节蛋白激酶1/2 核转录因子κB NF-κB抑制蛋白α 激活蛋白1 肿瘤坏死因子　出处：《暨南大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的:双歧杆菌的完整肽聚糖(Whole Peptidoglycan,WPG)是双歧杆菌发挥抗肿瘤、抗感染以及免疫赋活等多种生理功能的主要成分,它能激活机体免疫系统中的巨噬细胞,使其分泌多量的细胞毒性效应分子。目前对WPG激活巨噬细胞机制的认识还很肤浅。我们通过观察双歧双歧杆菌的WPG对大鼠腹腔巨噬细胞PKC→NF-κB通路、PKC→ERK1/2→AP-1通路及其对细胞因子TNF-α mRNA表达的影响,探讨双歧杆菌的WPG激活巨噬细胞的信号机制。方法:分离培养SD大鼠腹腔巨噬细胞,实验分为对照组、WPG刺激组和预孵组。(1)采用激光共聚焦显微镜技术观察巨噬细胞内NF-κB的核移位情况;(2)运用凝胶电泳迁移率检测技术(electrophoretic mobility shfit assay,EMSA)分析巨噬细胞NF-κB和AP-1的DNA结合活性的变化;(3)运用免疫蛋白质印迹(western blot)测定巨噬细胞磷酸化ERK1/2和IκBα的蛋白表达水平;(4)采用半定量逆转录多聚酶链反应(RT-PCR)技术检测巨噬细胞TNF-α mRNA的表达水平。结果:(1)正常大鼠腹腔巨噬细胞NF-κB位于胞浆内;WPG刺激巨噬细胞后,NF-κB被明显激活,移位进入胞核,应用EMSA检测到巨噬细胞NF-κB的DNA结合活性显著高于对照组(P0.01);应用激光共聚焦扫描显微镜观察到WPG刺激组巨噬细胞胞核NF-κB的阳性率明显高于对照组(P0.01);同时Western blot检测到胞质中IκBα的蛋白表达显著低于对照组(P0.01);但经PKC抑制剂Chelerythrine预孵巨噬细胞后,其NF-κB的移位和DNA结合活性及IκBα的降解较WPG刺激组明显减少(P0.01)。(2)未受WPG刺激的正常大鼠腹腔巨噬细胞ERK1/2处于低活性状态,给予WPG刺激巨噬细胞,其磷酸化ERK1/2的蛋白表达水平显著高于对照组(P0.01),但经PKC抑制剂Chelerythrine预孵巨噬细胞后,其磷酸化ERK1/2的蛋白表达水平明显低于WPG刺激组(P0.01)。(3)正常大鼠腹腔巨噬细胞AP-1位于胞浆内;WPG刺激巨噬细胞后,AP-1被明显激活,应用EMSA检测到巨噬细胞AP-1的DNA结合活性较对照组显著增高(P0.01);经ERK抑制剂PD98059预孵巨噬细胞后,其AP-1的DNA结合活性较WPG刺激组显著降低(P0.01)。(4)TNF-α mRNA在正常静息大鼠腹腔巨噬细胞内有低水平表达,WPG刺激组巨噬细胞TNF-α mRNA的表达显著高于对照组(P0.01);但经ERK抑制剂PD98059和NF-κB抑制剂NAC分别预孵巨噬细胞后,其TNF-α mRNA的转录水平均明显低于WPG
[Abstract]:Objective: peptidoglycan (Whole Peptidoglycan WPG) is Bifidobacterium antitumor, anti infection and immune components activating several physiological functions such as, it can activate the immune system in macrophages and cytotoxic effector molecules to produce a large amount of WPG. The current understanding of macrophage activation mechanism we have only a superficial. Through the observation of Bifidobacterium bifidum WPG on rat peritoneal macrophages of PKC NF-, B pathway, PKC - ERK1/2 - AP-1 pathway on cell factor TNF- alpha mRNA expression, signal mechanism of Bifidobacterium WPG activated macrophages.
Methods: the cultured SD rat peritoneal macrophages were divided into control group, WPG stimulation group and pretreatment group. (1) by laser confocal scanning microscope to observe the nuclear translocation of NF- in macrophages of kappa B; (2) using electrophoretic mobility detection technology (electrophoretic mobility shfit assay, EMSA) activity combined with the analysis of changes of macrophage NF- kappa B and AP-1 DNA; (3) using immune protein imprinted (Western blot) expression level determination of macrophage ERK1/2 phosphorylation and I kappa B alpha protein; (4) using semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression level of TNF- alpha mRNA macrophage technology.
Results: (1) kappa B normal rat peritoneal macrophages of NF- in cytoplasm; WPG stimulated macrophages, NF- kappa B was activated obviously, translocation into nucleus, the application of EMSA to detect macrophage NF- kappa B binding activity of DNA was significantly higher than the control group (P0.01); the positive rate of application of confocal laser scanning microscopy to WPG stimulation group macrophage nuclear NF- kappa B was significantly higher than the control group (P0.01); and Western blot detected in the cytoplasm of I kappa B alpha protein expression was significantly lower than the control group (P0.01); but the PKC inhibitor Chelerythrine were incubated with macrophages, the degradation of NF- kappa B translocation and binding activity of DNA and I kappa B alpha than WPG stimulation group decreased significantly (P0.01). (2) without stimulation of WPG in peritoneal macrophages of normal rats ERK1/2 in the low activity state, WPG stimulated macrophages, the expression level of the phosphorylated ERK1/2 protein was significantly higher than the control group (P0.01), but the PKC under Preparation of Chelerythrine pre incubated macrophages, the expression level of the phosphorylated ERK1/2 protein was significantly lower than that of WPG group (P0.01). (3) peritoneal macrophages of normal rats AP-1 in cytoplasm; WPG stimulated macrophages after AP-1 were significantly activated, application of EMSA to detect macrophage AP-1 binding activity of DNA was higher than that in the control group (P0.01); the ERK inhibitor PD98059 were incubated with macrophages, the AP-1 binding activity of DNA with WPG stimulation group decreased significantly (P0.01). (4) TNF- alpha mRNA expressed at a low level in normal resting rat peritoneal macrophages, the expression of WPG in macrophage TNF- alpha mRNA stimulation group was significantly higher than the control group (P0.01); but the ERK inhibitor PD98059 and NF- inhibitor NAC kappa B respectively were incubated with macrophages, the transcription level of TNF- alpha mRNA were significantly lower than that of WPG

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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