【作者】 宋卉；

【导师】 钱元恕；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2004， 博士

【摘要】 目的 观察头孢地嗪(Cefodizime，CDZ)对肺炎克雷伯杆菌(Klebsiella pneumoniae，Kle．p)引起的小鼠肺部炎症反应过程中，中性粒细胞(Polymorphonuclear neutrophil granulocytes，PMN)分泌细胞因子的变化，并探讨其与LPS-TLR-NFкB信号传导通路的关系。 方法 1．建立肺炎模型：经热灭活后的Kle．p 1x10~7CFU悬浮于50μ1PBS中，经鼻吸入法感染小鼠肺部。 2．给药方式及剂量：感染前96h开始给药，至接种细菌：将CDZ溶于PBS中30mg／kg，次／12h，皮下注射给药。 3．PMN的分离：感染3-24h后，不连续密度梯度法分离腹腔灌洗液中的PMN。 4．PMN表达的细胞因子种类强度分析：以含有96种细胞因子基汕头大学医学院博士研究生学位论文因表达谱的芯片检测感染前、后小鼠PMN表达细胞因子种类及强弱变化。5.尹材N.麦送匆分蘑时了W界a柳IL一叨总娜在感染的不同时间， 以Rl’- PCR方法检测PMN中TNF一a和IL一lp的mRNA表达情况; 以ELisA检测TNF一a、IL一lp分泌量的变化;以westem blot分析PMN内TNF一a、几一lp蛋白的表达。6.呼吸塌发翻启;以硝基四氮哇蓝还原实验(NBT test)检测PMN氧 依赖的呼吸爆发释放活性氧的情况。7.LP害.TLR.八子吮刀朦号传导通雳研穷{R:T- PCR方法检测PMN中 Toll样受体4(TLR4) mRNA表达情况;W七Stem blot分析PMN内 I一沼量的变化以反映其活性变化;凝胶电泳迁移率分析实验 (Eleetrophoretie mobility shift ass盯，EMSA)检测PMN.内核因子沼 (Nuelear factor一沼，NF一忍)的转录活性。结果小鼠PMN能表达多种细胞因子，热灭活Kle.p可诱导护什吓一a及IL一lp等基因表达增加。2.不N界。和IL一叨mR八月碑麦巡二卖COz对真彭嗬;(1)小鼠感染热灭活Kle.p后，PM囚中TNF一a和IL一1乃的mRNA表达 明显增强。TNF一Q表达在6h最强;几一IB表达在12h达到最高;24h 后二者表达均明显减弱但仍较对照组强。汕头大学医学院博士研究生学位论文(2)与模型组相比，CDZ促进感染3一6h后TNF一。和IL一1旦表达，但削弱了感染12一24h的TNF一Q和24h后的IL一1日mRNA表达。(3)CDZ 显影响对未感染小鼠PM[N的TNF一Q和IL一1 pmRNA表达未见明3.只UN道沙zN矛’-。柳IL一叨蛋户浦之友‘刀z刀真影时(l)在未感染小鼠PMN胞内TNF一。和IL一IB蛋白量较少，以Westem blot手段较难检出。Kle.p感染3h后，可见PMN胞内细胞因子的蛋白表达增加，6h后最高，12h后降低。(2)与模型组相比，CDz增强了感染小鼠感染3h后的TNF一a和3一1 Zh 的IL一1 6PMN胞内蛋白表达，而降低感染12h后的TNF一Q和24h后的IL一lp表达。(3)CDZ 显影响对未感染小鼠PMN胞内TNI;Q和IL一1日的蛋白表达未见明4.尸材N分泌劣里N声乙a、IL一1刀才平及CDZ训对真彭碱(l)小鼠感染热灭活Kle.p后，PMN分泌的TNF一。及IL一lp水平升高; TNF一。在感染后6h最高，24h后明显降低，但仍较对一照组高;而IL一lp在12h最高，24h后仍有较高分泌量。(2)与模型组相比，CDz增加了感染Kle.p小鼠3一6h后pMN的TNF-a和IL一lp分泌量、而降低感染12h后二者的分泌量。(3)CDZ对未感染小鼠PMN的T哪一a和IL一lp分泌未见明显影响。5一只UN时呼蟹廖发飞汕头大学医学院博士研究生学位论文(1)热灭活Kle.p感染促进小鼠PMN的呼吸爆发，以感染3h后最强， 此后渐低，但24h后仍较未感染组强。(2)与模型组相比，CDZ加强了小鼠感染3h后的呼吸爆发，12h后降低其呼吸爆发强度，24h后几乎回落至感染前水平。(3)CDZ对未感染小鼠的呼吸爆发无明显影响。6 .LP.多TLR.八子吮B存号撵.早涵歇(l)感染Kle.p后PMN中I破B水平明显降低，感染3h最低，6h后回 升，但在相同时间点上仍较未感染组低。与感染组相比，CDZ处理 可进一步降低感染6h内小鼠PMN中I一kB水平，而升高12一24h后 其水平;对未感染小鼠I一忍水平未见明显影响。(2)感染Kle.p刺激3h后TLR4的mRNA表达明显增加;6一24h后的 增加不明显。CDZ促进感染3一24h后的表达，3h后最明显。对未感染小鼠也有促进TLR4mRNA表达的作用。(3)在对照组小鼠PMN中，未见N’F一KB的激活，Kle.p感染后NF- KB的DNA结合活性明显加强，3h后最强，随后逐渐减弱。CDZ 明显加强感染6h内PMN的NF一KB活性，而在12h后则减弱其活 性。CDZ对未感染小鼠PMN的NF一KB的活性无明显影响。结论 CDZ对Kle.p感染诱导的小鼠PMN有双向免疫调节作用，其机制与调节TLR4表达和哪一沼的转录活性，从而影响TNF一a、IL一lp汕头大学医学院博士研究生学位论文等促炎因子的表达有关。更多还原

【Abstract】 Many antibiotics showed immunomodulatory effects in ways directly or indirectly. Indirect ways are mainly based on the antibacterial characterization of antibiotics, such as killing of bacteria, changing the intestinal flora and preventing bacteria making virulence factors to immune system. Some antibiotics have intrinsic antigenicity, such as beta-lactams and Rifampicin, and some of them may cause allergic reactions. Direct actions of antibiotics are mainly upon phagocytic functions including phagocytosis and killing, chemotaxis and lymphocyte activities. Among Cephalosporins, Cefodizime has been studied a lot. In rats and mice, Cefodizme was found to increase delayed hypersensitivity reactions, phagocytosis, lymphocytic responses to B cell mitogens and specific antigens and production of lysosomal enzymes and cytokines. However, the immunomodulating effects of CDZ and its mechanism on Klebsiella pneumoniae (Kle.p) stimulated neutrophils, the key cells in the non-specific (innate) defense in human and animal, were seldom investigated. This study was to explore the imrnunomodulating effects of CDZ on mice neutrophil and the influence of CDZ on the activity of LPS-TLR-NF-KB signal transduction pathway.METHODSMice were stimulated with Kle.p intranasally. CDZ was administrated subcutaneously at 30 mg/kg of body weight at 12-hour intervals, starting 96 hours before the inhalation of bacteria. Cytokine gene expression detected by DNA micro-array analysis after Kle.p stimulation. Effects of CDZ on the expression of TNF-a and IL-1 P of neutrophil evaluated by RT-PCR, ELISA and Western blot respectively. To elucidate the influence of CDZ on NF-KB signal transduction pathway, the expression of inhibitor of NF-KBa (I-KBa) was assayed by western blot. The transduction activity of NF-KB assayed by Electrophoretic Mobility Shift Assay (EMSA), the mRNA expression of toll like receptor 4 (TLR4), one of the most important receptor on LPS signal transduction pathway, detected by RT-PCR.RESULTSDNA micro-array analysis shows that neutrophil can express many cytokines and chemokines, such as TNF- a, IL-1 a, IL-1 P , IL-20, INF a, INF P , I K B, Fgf 16 and Fgf 17 etc. Kle. p stimulation increased cytokines expression in neutrophil, such as TNF- a , IL-1 P and IFN etc. The production and mRNA expression of TNF-a and IL-lp of neutrophils was enhanced by Kle.p stimulation in mice. CDZ treatment increased the production of cytokines and the degradation of I-KB in neutrophil during the first 3-6 hours after Kle.p stimulation. At the same time, NF-KBtransduction activity and TLR4 mRNA expression was promoted too. The production of cytokines and NF-KB DNA-binding activity were attenuated by CDZ 12-24 hours after Kle.p stimulation. The oxidative products of neutrophils in Kle.p stimulated mice was also enhanced by CDZ in the first 6 hours and attenuated 12 hours after Kle.p stimulation, which was also possibly related to the change of NF-KB activity.CONCLUSIONSCDZ shows dual immunomodulating effects on Kle.p stimulated neutrophil in mice. These effects may be due to the regulation on expression of TNF-a and IL-lp by affecting TLR4 mRNA expression and DNA binding activities of NF-KB through LPS-TLR4-NF-KB pathway.更多还原