本文關(guān)鍵詞： 雙歧桿菌 完整肽聚糖 巨噬細胞 蛋白激酶C 細胞外信號調(diào)節(jié)蛋白激酶1/2 核轉(zhuǎn)錄因子κB NF-κB抑制蛋白α 激活蛋白1 腫瘤壞死因子　出處：《暨南大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:雙歧桿菌的完整肽聚糖(Whole Peptidoglycan,WPG)是雙歧桿菌發(fā)揮抗腫瘤、抗感染以及免疫賦活等多種生理功能的主要成分,它能激活機體免疫系統(tǒng)中的巨噬細胞,使其分泌多量的細胞毒性效應(yīng)分子。目前對WPG激活巨噬細胞機制的認識還很膚淺。我們通過觀察雙歧雙歧桿菌的WPG對大鼠腹腔巨噬細胞PKC→NF-κB通路、PKC→ERK1/2→AP-1通路及其對細胞因子TNF-α mRNA表達的影響,探討雙歧桿菌的WPG激活巨噬細胞的信號機制。 方法:分離培養(yǎng)SD大鼠腹腔巨噬細胞,實驗分為對照組、WPG刺激組和預(yù)孵組。(1)采用激光共聚焦顯微鏡技術(shù)觀察巨噬細胞內(nèi)NF-κB的核移位情況;(2)運用凝膠電泳遷移率檢測技術(shù)(electrophoretic mobility shfit assay,EMSA)分析巨噬細胞NF-κB和AP-1的DNA結(jié)合活性的變化;(3)運用免疫蛋白質(zhì)印跡(western blot)測定巨噬細胞磷酸化ERK1/2和IκBα的蛋白表達水平;(4)采用半定量逆轉(zhuǎn)錄多聚酶鏈反應(yīng)(RT-PCR)技術(shù)檢測巨噬細胞TNF-α mRNA的表達水平。 結(jié)果:(1)正常大鼠腹腔巨噬細胞NF-κB位于胞漿內(nèi);WPG刺激巨噬細胞后,NF-κB被明顯激活,移位進入胞核,應(yīng)用EMSA檢測到巨噬細胞NF-κB的DNA結(jié)合活性顯著高于對照組(P0.01);應(yīng)用激光共聚焦掃描顯微鏡觀察到WPG刺激組巨噬細胞胞核NF-κB的陽性率明顯高于對照組(P0.01);同時Western blot檢測到胞質(zhì)中IκBα的蛋白表達顯著低于對照組(P0.01);但經(jīng)PKC抑制劑Chelerythrine預(yù)孵巨噬細胞后,其NF-κB的移位和DNA結(jié)合活性及IκBα的降解較WPG刺激組明顯減少(P0.01)。(2)未受WPG刺激的正常大鼠腹腔巨噬細胞ERK1/2處于低活性狀態(tài),給予WPG刺激巨噬細胞,其磷酸化ERK1/2的蛋白表達水平顯著高于對照組(P0.01),但經(jīng)PKC抑制劑Chelerythrine預(yù)孵巨噬細胞后,其磷酸化ERK1/2的蛋白表達水平明顯低于WPG刺激組(P0.01)。(3)正常大鼠腹腔巨噬細胞AP-1位于胞漿內(nèi);WPG刺激巨噬細胞后,AP-1被明顯激活,應(yīng)用EMSA檢測到巨噬細胞AP-1的DNA結(jié)合活性較對照組顯著增高(P0.01);經(jīng)ERK抑制劑PD98059預(yù)孵巨噬細胞后,其AP-1的DNA結(jié)合活性較WPG刺激組顯著降低(P0.01)。(4)TNF-α mRNA在正常靜息大鼠腹腔巨噬細胞內(nèi)有低水平表達,WPG刺激組巨噬細胞TNF-α mRNA的表達顯著高于對照組(P0.01);但經(jīng)ERK抑制劑PD98059和NF-κB抑制劑NAC分別預(yù)孵巨噬細胞后,其TNF-α mRNA的轉(zhuǎn)錄水平均明顯低于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

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

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