【作者】 刘俊文；

【导师】 肖献忠；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 脓毒症(sepsis)是由感染引起的全身炎症反应综合征(systemicinflammatory response syndrome,SIRS)。虽然其发生机制尚未完全阐明,但是革兰氏阴性细菌胞壁上的脂多糖(lipopolysacchride,LPS)即内毒素启动体内免疫系统在脓毒症发病中的重要性已受到广泛关注。LPS进入细胞内可激活多条信号转导通路,通过直接或间接的方式激活炎症相关转录因子,从而导致大量下游炎症介质基因的过度表达。Kruppel样因子4(Kruppel-like factor 4,KLF4)是Sp1/KLF锌指转录因子家族的成员之一。KLF4能够与下游基因启动子区的GC盒,CACCC盒和基础转录元件(basic transcription elements)三种结合元件相结合,从而直接调控这些基因的表达。通过对下游基因表达的调控,KLF4在正常细胞的增殖和分化、胚胎发育以及肿瘤的发生发展过程中发挥重要作用。但是在LPS作用下KLF4呈何种表达模式,KLF4是否对LPS所致的炎症介质基因表达具有调控作用,以及KLF4如何发挥调控作用等问题,目前仍不清楚。本实验室近年采用cDNA微阵列检测了内毒素休克小鼠肺组织基因表达谱的变化,发现KLF4基因在注射内毒素2小时的小鼠肺组织中表达上调;在注射内毒素20小时后,表达水平进一步增高,达正常水平的10倍以上。以上结果提示,KLF4可能是一个在炎症反应中起重要作用的转录因子,它的表达改变可能参与了LPS诱导的炎症介质基因的表达调控。阐明KLF4在调控LPS所致的全身炎症反应中的生物学功能,对于揭示脓毒症的发生机制,进一步探讨其有效防治措施,可能具有重要意义。为了观察KLF4在LPS作用下的表达模式,本研究第一部分采用人THP-1单核细胞、人U937单核巨噬细胞和小鼠RAW264.7巨噬细胞作为研究对象,采用RT-PCR和Western blot分别于mRNA和蛋白质水平检测KLF4在内毒素反应中的表达,发现LPS导致多种单核巨噬细胞中KLF4的表达显著增加,并呈时间与剂量依赖模式;在RAW264.7巨噬细胞中KLF4的表达在LPS刺激的早期(1-2 h)即明显增加,并且持续至晚期(24 h以后)。为了观察KLF4是否对炎症介质基因具有调控作用,本研究第二部分首先构建了KLF4过表达的RAW264.7细胞株,并采用反义寡核苷酸技术建立了内源性KLF4表达抑制的模型;根据生物信息学对炎症介质基因启动子区的分析及RT-PCR的筛选结果,选取了两个具有代表性的早期炎症介质基因IL-1β和IL-10,观察了KLF4对这两个基因表达及释放的影响,并对其调控机制进行了探讨,获得了以下实验结果:(1)KLF4对LPS刺激下IL-1β和IL-10 mRNA表达水平的影响。正常状态下,细胞的IL-1βmRNA表达水平较低;LPS刺激之后,IL-1β的mRNA表达明显升高并在2 h时达到高峰,而KLF4过表达明显抑制了LPS诱导的IL-1βmRNA的表达;KLF4的过表达使IL-10 mRNA的基础表达增高,而LPS刺激之后,IL-10 mRNA的诱导表达显著高于空载体组。(2)KLF4对LPS刺激下IL-1β和IL-10蛋白质表达水平的影响。KLF4过表达能够抑制LPS诱导的IL-1β释放,促进IL-10的释放;而KLF4内源性抑制则促进IL-1β的释放,抑制IL-10的诱导释放。(3)LPS促进KLF4与IL-1β和IL-10基因启动子区的结合活性。在正常状态下KLF4能够与IL-1β基因启动子区-35～-6bp和IL-10基因启动子区-244～-215bp结合,LPS能够促进此结合活性的增强。(4)KLF4对LPS刺激下IL-1β和IL-10基因启动子转录活性的影响。KLF4能够抑制正常状态下IL-1β基因启动子的转录活性,并明显抑制LPS所致的IL-1β基因启动子转录活性的增强;KLF4能够激活正常状态下IL-10基因启动子的转录活性,促进LPS所致的IL-10基因启动子转录活性的增强。由于KLF4的表达水平在LPS刺激的晚期(24 h以后)仍然升高明显,这提示KLF4可能对晚期炎症介质也具有调控作用。本文第三部分选取了晚期炎症介质HMGB1进行研究。结果发现:(1)KLF4能够促进HMGB1的基础表达,并促进LPS刺激下的HMGB1表达、移位及释放。(2)KLF4可以与HMGB1基因启动子区-736～-707bp和-1317～-1304bp结合,LPS能够促进其结合活性。上述结果表明,KLF4通过抑制早期促炎介质IL-1β的表达与释放及促进早期抗炎介质IL-10的表达与释放可能在LPS所致的SIRS早期发挥抑炎作用;但通过促进晚期促炎介质HMGB1的表达、移位及释放可能在LPS所致的SIRS晚期发挥促炎作用。综上所述,本研究发现了KLF4的一个新功能,即能够通过与某些炎症基因启动子区的KLF4结合元件相结合来直接调控这些基因的表达,并影响其释放。目前认为脓毒症发病机制的本质是失控性炎症反应,转录因子对各种炎症介质的调控起到了非常重要的作用。KLF4对LPS所致炎症反应的双向调节作用,为揭示脓毒症的发病机制提供了新的实验依据,也为深入探讨KLF4的生物学功能和脓毒症的防治提供了新的线索。更多还原

【Abstract】 Sepsis is the systemic inflammatory response syndrome(SIRS) induced by infection.It has been emphasized that lipopolysaechride(LPS) plays important role in the pathogenesis of sepsis by initiating the immune system.Many studies have demonstrated that LPS mediates activation of multiple signal-transduction pathways and some inflammation-related transcriptional factors,and then promotes the expression of multiple target inflammatory genes.However,the particular mechanisms of sepsis are still obscure.Kruppel-like factor 4(KLF4),a member of Sp1/KLF zinc-finger transcriptional factor family,binds to the binding sites including GC box, CACCC box and basic transcription elements on the promoters of target genes,to regulate the expression of target genes directly.By regulating the expression of the target genes,KLF4 plays important roles in cellular proliferation and differentiation,embryogenesis and the development of carcinoma.It is not delineated on the expression pattem of KLF4 under LPS treatment,and the roles and mechanisms of KLF4 in regulating the expression of inflammatory genes.Our previously research from cDNA microarray assays revealed that the expression level of KLF4 was up-regulated in lung tissues at 2 h,and further increased up to more than 10 times of the normal level at 20 h after LPS injection.These results indicate that KLF4 may be an important transcriptional factor participating in the expression regulation of inflammatory mediator genes induced by LPS.To verify the hypothesis mentioned above,we first investigated the expression pattern of KLF4 in LPS-mediated inflammatory cell models in human THP-1 monoeytes,human U937 monocytes/macrophages and mouse RAW264.7 macrophages by RT-PCR and Western blot.The results showed that the expression of KLF4 was up-regulated obviously in all the three kinds of monoeytes/maerophages in a time and dose-dependent manner after exposure to LPS.Meanwhile,the expression of KLF4 in RAW264.7 maerophages was increased significantly at the early phase (within 1-2 h)and further increased up to the late phase(after 24 h)after LPS stimulation.In order to understand the roles of KLF4 in regulating the expression of inflammatory mediator genes,we first overexpressed mouse KLF4 cDNA gene(pcDNA3.1-KLF4)into RAW264.7 macrophages by gene transfection and G418 screening,and inhibited the expression of KLF4 gene in RAW264.7 macrophages by transfection of KLF4 antisense oligonucleotides.Then,according to the screening results from bioinformatics and RT-PCR,we selected IL-1βand IL-10,two representative inflammatory mediator genes activated at early phase of inflammation,to investigate the effect of KLF4 on their expression and release under normal condition and LPS treatment.The results showed that,overexpression of KLF4 could inhibit the expression and release of IL-1βinduced by LPS,but promote the expression and release of IL-10 under the normal condition and after LPS stimulation;on the other hand, the inhibition of KLF4 by antisense oligonucleotides could promote the expression and release of IL-1β,and inhibit the expression and release of IL-10 under normal condition and after LPS stimulation.The results from EMSA demonstrated that KLF4 could bind to the KLF4 binding sites on the promoters of IL-1βand IL-10 genes,and the results from luciferase reporter gene assay demonstrated that KLF4 could inhibit the transcriptional activity of IL-1βgene and promote the transcriptional activity of IL- 10 gene.According to the up-regulation of KLF4 at the late phase after LPS stimulation,we predicted that KLF4 might also regulate the inflammatory mediator gene activated at the late phase.Therefore,we investigated the effect of KLF4 on the expression and release of HMGB1,an important inflammatory mediator gene released at the late phase of inflammation. The results showed that the levels of HMGB1 mRNA and protein were up-regulated under normal condition after KLF4 over-expression;after LPS stimulation,KLF4 overexpression promoted the expression, translocation and release of HMGB1.EMSA showed that KLF4 could bind to the sites at -736～-707 bp and -1317～-1304 bp on the HMGB1 promoter,and the binding activity was promoted by LPS stimulation.It is indicated that KLF4 plays a bidirectional role in the regulation of inflammation induced by LPS.It may play anti-inflammatory role at the early phase of SIRS induced by LPS by inhibiting the expression and release of early pro-inflammatory mediator IL-1βand promoting the expression and release of early anti-inflammatory mediator IL-10;and may play pro-inflammatory role at the late phase of SIRS induced by LPS by increasing the expression,translocation and release of late pro-inflammatory mediator HMGB1.Taken together,the present studies suggest a novel function of KLF4, that is,KLF4 can bind to the KLF4 binding sites on the promoters of inflammatory mediator genes including IL-1β,IL-10 and HMGB1 to regulate the expression and release of these genes.The bidirectional regulating effects of KLF4 on early or late inflammatory mediators provide novel insights into the pathogenesis of sepsis,and also new clues and information for the studies on the biological functions of KLF4,and the prevention and treatment of sepsis.更多还原