论文标题:不可分型流感嗜血杆菌（NTHI）致宿主细胞炎症反应的分子发病机制研究
The Molecular Mechanisms of Non-typeable Haemophilus Influenza (NTHI) Induced Inflammatory Response in Host Cells
论文作者
论文导师 李兰娟,论文学位 博士,论文专业 内科学
论文单位 浙江大学,点击次数 117,论文页数 86页File Size7250K
2008-06-01论文网 http://www.lw23.com/lunwen_235901037/
Non-typeable Hemophilus influenza (NTHI);; mitogen activated protein kinase(MAPK);; toll-like receptor(TLR);; monocyte;Non-typeable Hemophilus influenza (NTHI);; mitogen activated protein kinase (MAPK);; NF-κB;; respiratory epithelial cells;; soluble cytoplasmic fractions (SCF);; transmission electron microscope (TEM);Non-typeable Hemophilus influenza (NTHI);; mitogen activated protein kinase(MAPK);; NF-κB;; respiratory epithelial cells;; Cycloxygenase (COX);; Prostaglandin E (PGE2);; Toll-like receptor (TLR);; gene knocked-out mice
第一部分:p38 MAPK在NTHI致人单核细胞炎症反应中的作用 目的:通过不可分型流感嗜血杆菌与单核细胞相互作用,研究丝裂原活化的蛋白激酶(mitogen activated protein kinase,MAPK)信号传导通路在不可分型流感嗜血杆菌(nontypeable haemophilus influenza,NTHI)致人体免疫细胞炎症反应的作用。方法:NTHI为浙江大学医学院附属第二医院临床分离株,经血清学和16sRNA测序证实,外周血单核细胞分离自健康成年人的静脉血。NTHI与单核细胞共培养,1 h、4 h后收集细胞用Western Blot检测p38、p44/42-MAPK的磷酸化;16 h后用流式细胞仪检测细胞表面TLR4的表达。预先用p38-MAPK抑制剂(SB203580)和p44/42-MAPK抑制剂(UO126)与单核细胞共孵育1 h,然后加入NTHI(感染复数:200),分别在4 h、16 h后收集上清,以酶联免疫吸附法检测肿瘤坏死因子α(TNF-α)的水平。结果:NTHI能迅速地诱导p38,p44/42-MAPK通路的磷酸化,并至少持续到刺激后4 h。与未加细菌组比较,NTHI刺激16h后单核细胞表面TLR4受体表达明显增加,上清中TNF-α增加(p＜0.05)。与细菌攻击组比较,阻断p38-MAPK,而不是阻断p44/42-MAPK通路,能显著地降低单核细胞分泌TNF-α(p＜0.05)。结论:TLR4可能参与了NTHI致单核细胞炎症反应;p38-MAPK是NTHI诱导单核细胞炎症反应的关键信号分子。 第二部分:NTHI致呼吸道上皮细胞炎症反应的分子机制研究 目的:通过建立呼吸道上皮细胞株的体外NTHI感染模型,确认NTHI在细胞内的存活,观察NTHI及其组分(可溶性胞质成分SCF、外膜蛋白EP)对呼吸道上皮细胞MAPK信号传导通路和后续的前炎症因子表达的影响,并应用特异性ERK-MAPK抑制剂U0126、p38 MAPK抑制剂SB203580特异性阻断下游MAPK信号传导通路,观察它们对呼吸道上皮细胞分泌前炎症因子表达的影响。方法:NTHI系临床分离株,经常规和基因测序鉴定。NTHI与气道上皮细胞共培养,透射电镜观察A549细胞内定植,12h后Triton破膜,再行培养证实细菌存活;Westernblot检测A549及BEAS-2B细胞p-p38和p-ERK MAPK蛋白的表达;超声打碎细胞,超速离心分离NTHI可溶性胞质成分和外膜蛋白,流式细胞仪测定A549细胞NF-κB p65的表达;ELISA测定A549和BEAS-2B细胞IL-8的表达。结果:NTHI在感染A549细胞4h后即有大量NTHI进入胞内,且细胞内的NTHI数目与感染时间和细菌数目成正比;用Triton X-100破膜,对胞内细菌行体外培养,在巧克力平板上过夜后即见NTHI生长。Western blot显示:NTHI刺激15min-30min后,A549细胞内的p-p38和p-ERK MAPK表达开始增加;NTHI刺激15min-30min后,BEAS-2B细胞内的p-p38、p-ERK MAPK表达开始增加。流式细胞法显示:NTHI和SCF、EP刺激4h后,A549细胞内NF-κB p65的表达较培养基组明显增强;与培养基对照比较,NTHI、SCF、EP组均明显增强,其中完整NTHI作用最强。A549在NTHI刺激后IL-8分泌增加,可被SB203580抑制,但不被UO126抑制。BEAS-2B细胞在NTHI刺激后IL-8分泌增加,可部分被两种MAPK抑制剂所抑制。结论:NTHI可以进入A549细胞并存活,是兼性的胞内菌;NTHI及其菌体成分对肺上皮细胞均有较强的致炎作用,完整细菌的作用最强;p38MAPK、ERK MAPK和NF-κB均参与NTHI致气道上皮炎症反应,但MAPK参与NTHI致肺泡上皮细胞和支气管上皮细胞的炎症反应时的通路有所不同,p38可能是主要的信号传导通路。 第三部分:NTHI感染对气道上皮细胞AA-COX-PGE2代谢途径的影响和分子调控机制 目的:通过建立NTHI感染气道上皮细胞的体外模型和小鼠的感染模型,研究NTHI诱导气道上皮细胞和肺组织COX2和PGE2的表达,揭示Toll样受体在NTHI致气道上皮细胞和肺组织炎症反应中的作用,同时阐明p38MAPK和NF-κB两条信号传导通路的作用及两者之间的关系。方法:用不同浓度的NTHI感染A549细胞以建立细胞体外感染模型。Western blot检测p38MAPK的磷酸化,电泳迁移率试验测定NF-κB DNA结合活力,COX-1、COX-2 mRNA和PGE2蛋白的表达分别采用逆转录聚合酶链反应和酶联免疫吸附试验。用高表达TLR2和TLR4的HEK293细胞试验以及用TLR2、TLR4基因敲除小鼠体内试验,研究TLR2、TLR4在COX-2表达中的作用。另外,用特异性分子抑制剂阻断p38MAPK和NF-κB来揭示两条信号传导通路的作用。结果:在A549细胞NTHI诱导COX-2 mRNA表达呈剂量相关性,但不影响COX-1 mRNA的表达。NTHI感染引起的PGE2表达增高可以被特异性的COX-2抑制剂所抑制,但不被COX-1抑制剂所抑制。NTHI诱导的COX-2表达在上皮细胞的体外试验和肺组织的体内试验中都是通过TLR2介导的。NTHI可以诱导p38 MAPK磷酸化和NF-κB DNA结合活力的上调,COX-2和后续PGE2可以被p38 MAPK及NF-κB特异性抑制剂所阻断;但是NTHI诱导的NF-κB DNA结合活力的上调不受p38 MAPK抑制剂的影响。结论:NTHI感染气道上皮细胞是通过COX-2而非COX-1引起PGE2表达增高;无论是体外还是体内,TLR2是介导NTHI诱导COX-2和PG E2表达的主要表面受体;NTHI诱导COX-2和PGE2表达是通过p38 MAPK及NF-κB两条信号传导通路进行调节的,两者之间是相对独立的。
PART 1:The role of p-38 MAPK pathway in the NTHI induced inflammatory response in human monocytes Objective This study is to elucidate the role of MAPK pathway in the inflammatory reaction induced by NTHI.Methods NTHI was from clinical isolates which was identified by serum agglutination test and 16s rRNA.The peripheral monocytes were isolated from healthy donator.NTHI was cultured with monocytes and monocytes were harvested 1 and 4h after infection.MAPK phosphorylation was detected by Western Blot.Expression of toll-like receptor 4(TLR4)was detected by flow cytometry in 16h.Monocytes were incubated with MAPK inhibitors for 1h and then infected with different concentrations of NTHI for 4 and 16h,respectively.TNF-αlevel was determined by ELISA.Results Different concentrations of NTHI can rapidly induce p38 MAPK and p44/42 MAPK phosphorylation which continued for at least 4 h.Expression of TLR4 and TNF-αwas markedly up-regulated compared with the control group.Compared with NTHI stimulation,blockage of p38 MAPK,but not p44/42 MAPK,can remarkably decrease TNF-αsecretion from monocytes. Conclusion p38 MAPK is the key signal molecule in the inflammatory reactions of monocytes induced by NTHI. PART 2:The molecular mechanisms of Nontypeable Haemophilus influenzae induced inflammatory response in respiratory epithelial cells Objective:Nontypeable Haemophilus influenzae(NTHI)was traditionally considered as an extracellular pathogen in respiratory infections.In this study,we established a model of infected cells to confirm NTHI was also a facultatively intracellular pathogen.Mitogen activated protein kinases(MAPK)are the important signal transduction pathway in mediating inflammatory response induced by NTHI in respiratory epithelium.In this study,we elucidate the impact of NTHI,and its SCF and EP on the expression of MAPK and subsequent proinflammatory mediators. Methods:NTHI was a clinical isolate and identified by 16s rRNA sequencing.TEM and bacterial culture were used to determine the intracellular conlonization of NTHI in an epithelial cell line A549.Expression of p-p38 MAPK and p-ERK MAPK were detected by Western blot.SCF and EP of NTHI were separated by ultrasonic breaking and super-centrifugalization.FCAS was employed in analysis of NF-κB p65 expression.IL-8 levels were measured by enzyme linked immunosorbent assay (ELISA).In addition,the role of MAPK on IL-8 expression induced by NTHI was investigated by using specific MAPK inhibitors. Results:The number of NTHI penetrated in A549 cells increased in a time-dependant manner 4h-24h after infection.Culture of NTHI on Chocolate plate 12h after infection confirmed the bacteria still survive in A549 cells.Western blot analysis showed that p-p38 MAPK and p-ERK MAPK expression increased in A549 and BEAS-2B cells after NTHI stimulation.FCAS demonstrated that NF-κB p65 expression was up-regulated 4h after stimulation of NTHI,SCF and EP,and NTHI was the strongest stimulator among these stimulators.Enhanced IL-8 expression induced by NTHI in A549 can be inhibited by SB203580,but not UO126,whereas increased IL-8 expression induced by NTHI in BEAS-2B can be markedly inhibited by both SB203580 and UO126. Conclusion:As a facultatively intracellular pathogen,NTHI has the potential to penetrate and survive in alveolar epithelial cells.NTHI,SCF and EP were all strong inducer of inflammatory response in respiratory epithelium,and the intact bacterium was the strongest one.p38 MAPK,ERK MAPK and NF-κB all participated in NTHI induced inflammatory response in respiratory epithelial cells,and p38 MAPK may be the main signal transduction molecular. PART 3:Nontypeable Haemophilus influenzae induces COX-2 and PGE2 expression in lung epithelial cells via activation of p38 MAPK and NF-kappa B Background:Nontypeable Haemophilus influenzae(NTHI)is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary diseases,but its molecular interaction with human lung epithelial cells remains unclear.Herein,we tested that the hypothesis that NTHI induces the expression of cyclooxygenase(COX)-2 and prostaglandin E2(PGE2)via activation of p38 mitogen-activated protein kinase(MAPK)and nuclear factor(NF)-kappa B in pulmonary alveolar epithelial cells. Methods:Human alveolar epithelial A549 cells were infected with different concentrations of NTHI.The phosphorylation of p38 MAPK was detected by Western blot analysis,the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay(EMSA),and the expressions of COX-1 and 2 mRNA and POE2 protein were measured by reverse transcription-polymerase chain reaction(RT-PCR)and enzyme linked immunosorbent assay(ELISA),respectively. The roles of Toll-like receptor(TLR)2 and TLR4,well known NTHI recognizing receptor in lung epithelial cell and gram-negative bacteria receptor,respectively,on the NTHI-induced COX-2 expression were investigated in the HEK293 overexpressing TLR2 and TLR4 in vitro and in the mouse model of NTHI-induced pneumonia by using TLR2 and TLR4 knock-out mice in vivo.In addition,the role of p38 MAPK and NF-kappa B on the NTHI-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors. Results:NTHI induced COX-2 mRNA expression in a dose-dependent manner,but not COX-1,mRNA expression in A549 cells.The enhanced expression of PGE2 by NTHI infection was significantly decreased by pre-treatment of COX-2 specific inhibitor,but not by COX-1 inhibitor.NTHI induced COX-2 expression was mediated by TLR2 in the epithelial cell in vitro and in the lungs of mice in vivo. NTHI induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B.Moreover,the expressions of COX-2 and PGE2 were significantly inhibited by specific inhibitors of p38 MAPK and NF-κB.However,NTHI-induced DNA binding activity of NF-κB was not affected by the inhibition of p38 MAPK. Conclusion:NTHI induces COX-2 and PGE2 expression in a p38 MAPK and NF-κB-dependent manner through TLR2 in lung epithelial cells in vitro and lung tissues in vivo.The full understanding of the role of endogenous anti-inflammatory PGE2 and its regulation will bring new insight to the resolution of inflammation in pulmonary bacterial infections.
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