【作者】 林玲；

【导师】 孙乐栋；

【作者基本信息】 南方医科大学 ， 皮肤与性病学， 2010， 硕士

【摘要】 研究背景和目的：痤疮是一种累及毛囊皮脂腺的慢性炎症性疾病,其皮疹表现多样,好发于面颊、额部及胸背部等富含皮脂腺的部分。随着人们生活节奏的加快,痤疮的发病率也越来越高,部分重型痤疮病程慢性,易反复发作,少数患者病情可迁延十余年之久,皮疹消退后易遗留有碍于面部外观的瘢痕等,往往给患者带来较严重的精神负担。痤疮的病因较多,目前认为主要与下列4种因素有关：皮脂分泌过多,毛囊口的角化过度,痤疮丙酸杆菌的定植与侵袭,炎症反应。其中炎症反应是痤疮发病机制中的重要组成部分。痤疮丙酸杆菌是首要因素。痤疮丙酸杆菌能分泌许多前炎症介质,诱导局部炎症反应和机体对其的免疫反应,但是对痤疮丙酸杆菌引起的炎症机制不清。最近研究证实痤疮丙酸杆菌通过TLR(Toll-like-receptors, TLRs)依赖途经激活单核细胞,从而释放细胞因子引起炎症。微生物入侵机体后,机体的天然免疫系统首先对其进行识别并加以清除。天然免疫被认为是抵御微生物侵袭的第一道防线。寄生于毛囊内的痤疮丙酸杆菌,可被免疫细胞的天然免疫模式识别受体识别,通过启动天然免疫应答进而启动适应性免疫应答,诱导痤疮的炎症过程。Toll样受体(Toll-like receptor, TLR)是近年来新发现的一个介导天然免疫的古老家族。该受体的发现最早缘于对果蝇Toll蛋白的研究。Toll与配体结合能激活胞浆内的转录因子,诱导针对微生物病原体的宿主防御反应。在人类,也存在与之高度同源的功能相似的跨膜受体,称之为Toll样受体。该受体能有选择地识别细菌中的菌壁成分如脂多糖(lipopolysaccharide, LPS)、磷壁酸等。Toll样受体与之结合,并启动胞内一系列细胞因子的转录反应以清除病原微生物。因此该受体对机体的抗感染免疫意义重大。目前人Toll样受体家族中已确认的成员有10个(TLR1-TLR10),其中不同的亚型识别不同细菌菌壁中的病原相关分子模式(pathgon-associated molecular patterns, PAMPs)。TLR识别病原微生物表面相应的配体后,主要通过MyD88-IRAK-TRAF6激酶途径,活化下游核因子-κB (nuclear factor kappa B, NF-κB)和丝裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPK)信号途径,导致多种细胞因子、趋化因子、协同刺激因子及非特异性抗微生物肽的释放、多种免疫细胞的成熟及活化而介导针对该微生物的天然及获得性免疫应答,以清除入侵的病原微生物。TLR不仅能激活天然免疫,而且也为激活获得性免疫提供共刺激信号,因而在天然免疫与获得性免疫中起重要作用,是联系两者的桥梁。TLR2作为重要炎性受体,介导炎性损伤,其机制目前已部分明了。一系列实验证明,痤疮丙酸杆菌诱导机体的免疫应答主要是通过TLR2来介导的。痤疮皮损内可见TLR2表达,特别在毛囊及毛囊周围,且TLR2阳性细胞的数量随着皮损持续时间的延长而增加。用不同浓度超声破碎的痤疮丙酸杆菌来刺激原代培养的正常人单核细胞,可见有IL-12及IL-8呈剂量依赖式产生,此现象可被抗-TLR2中和抗体所阻断。Kim等证实痤疮丙酸杆菌通过TLR2依赖途径激活单核细胞,并且还发现在痤疮损害中单核细胞有TLR2表达,而淋巴细胞则不表达。Mochizuki H等在研究中证实,痤疮丙酸杆菌激活单核细胞释放细胞因子是通过TLR2介导。配体是痤疮丙酸杆菌细胞壁的肽聚糖。痤疮丙酸杆菌能够激活转染有TLR2的转录因子NF-κB途径,细菌通过PAMPs激发先天免疫,而PAMPs要与模式识别受体(phosphate buffer solution, PRRs)结合起作用。首先痤疮丙酸杆菌细胞壁的肽聚糖被TLR2所识别,在CD14的参与下,与TLR6形成异二聚体,并通过TIR区域(TLR/IL—1R)传递信号和激发下游效应。在TIRE与接头蛋白MYD88的羧基端的TLR受体域结合。而接头蛋白MYD88通过氨基端的死亡结构域与白介素1受体相关激酶(IL-1 receptor-associated kinase, IRAK)的氨基端的死亡结构域接合。二者作用导致IRAK自身的磷酸化而激活,继而激活肿瘤坏死因子-6(TRAF-6)通过使得IRAK和IRAK2连接于MAPK家族中的NF-KB诱导激酶(NF-κB inducing kinase, NF-κB)激活,进一步活化NF-κB使得其转位到细胞核,从而启动细胞因子如IL-8、TNF-α等参与免疫及炎症反应。这些细胞因子是痤疮出现炎性皮损的主要原因。本实验通过研究痤疮患者外周血CD14+单核细胞Toll样受体2(Toll-like receptor2, TLR2)的表达及其与IL-8、TNF-α浓度的相关性,初步探讨TLR2在痤疮发病中的作用。方法：本实验研究对象为2009年1月～2009年6月在我所门诊就治的痤疮患者共30例,根据《临床皮肤病学》的诊断标准确诊,且符合Pillsbury分类法中的Ⅲ级及Ⅳ级痤疮,所有痤疮患者近2个月内未系统服用维A酸、类固醇激素、免疫抑制剂等药物,2周内未使用任何外用药物。另取20例正常人做为对照组,均为本所职工或健康献血者,无免疫性疾病及系统性疾病。以上病例及正常人对照组均排除有合并其它病毒、细菌等感染或其它重大疾病。正常人对照组与痤疮患者组在性别及年龄上差异无统计学意义。在消毒条件下,自肘静脉抽取静脉血5mL。其中3ml全血用来进行流式细胞术检测。另2ml血液置于消毒的试管中,低温离心机离心10分钟(2500转/分),小心吸取其上层血清置-20℃冰箱中保存备用。采用多色免疫荧光流式细胞仪技术,观察这30例痤疮患者和20例正常对照外周血CD14+单核细胞上TLR2分子的表达率和平均荧光强度；采用双抗体夹心酶联免疫吸附法(ELISA)检测血清IL-8、TNF-α的浓度。病例组与正常对照组均数差异显著性检验采用t检验；TLR与IL-8、TNF-α的相关系数r计算采用Pearson分析。结果：1、痤疮患者外周血CD14+单核细胞TLR2的表达：痤疮患者外周血CD14+单核细胞TLR2的表达率平均为(50.88±3.14)%,平均荧光强度(MIF)为(2.44±0.34),较正常人对照组均显著增高(P<0.01)。2、痤疮患者外周血IL-8、TNF-α含量显著增高,明显高于正常人对照组,差异有统计学意义(P<0.01)。运用相关性检验分析显示TLR2的表达与血清IL-8、TNF-α的浓度呈正相关,TLR2表达百分率与IL-8、TNF-α相关系数分别为：0.966、0.739(P<0.01)。结论：痤疮患者TLR2在外周血CD14+单核细胞阳性率与平均荧光强度均高于正常人群,且痤疮患者TLR2的表达与血清IL-8、TNF-α浓度有显著相关性。结果提示TLR2及其介导的天然免疫应答在痤疮发病机制中起重要作用,其机制可能是通过上调TLR2的表达促使炎症因子产生和分泌而介导痤疮的发病。更多还原

【Abstract】 Background and Objective:Acne is a chronic inflammable disease involving the hair follicle and sebaceous gland,which usually was seen in the cheeks, forehead,chest and back riched in sebaceous glands. Recently, the incidence of Acne has increased,and some serious usually became chronic, recurrent, even the conditions has persistented more than ten years. The patients thus suffered from stress when having a facial scars appearance after the rash disppeared.There many factors involved in the mechanism of Acne, mainly four factors may explain the reasons, excessive sebum secretion, follicular hyperkeratosis, propionibacterium acne colonization and invasion, inflammation, and propionibacterium acne was considered as a major element during the duration of Acne,while inflammation as an important part of these factors. Propionibacterium acne secretes many proinflammatory cytokines, induces inflammatory response and the immune response, but the interacitions between these factors did not ascertain clearly. Recent studies have confirmed that Propionibacterium acne actives monocytes via TLR(Toll—like receptors, TLRs), releases cytokines and induce inflammatory response.After entering the body, the microbes were recoganized and cleared firstly by innate immune system.Innate immune system was considered as a first line of defense against microbial invasion. Propionibacterium within the hair follicle was recgonized by innate immune pattern recognition receptor from immune cells.Adaptive immune response was initiated seconderly after innate immune system actived, induced inflammation.It is now known that the innate immune system was considered as a first line of defense against conserved molecular patterns of components of microorganisms directly. Propionibacterium within the hair follicle was recgonized by innate immune pattern recognition receptor from immune cells. Adaptive immune response was initiated seconderly after innate immune system activated, induced inflammation. Toll-like receptor (Toll-like receptor, TLR) are the ancient family of innate immune system which has been discovered in recent years. Study on Toll protein of Drosophila found that Toll and it’s ligand binding can activate the transcription factor within the cytoplasm,induce defense response against pathogen microorganisms, In humans, there are also highly homologous transmembrane receptors being as similar functions as Toll protein of Drosophila, called Toll-like receptor, which can selectively identify patterns of components of bacteria such as LPS, teichoic acid and so on. Toll receptor bind them, and results in initiation of several signaling cascades, which eventually result in transcription reaction to remove pathogenic microorganisms. TLRs can also be classified into two groups based upon cellular location,and include 10 members totally. Different subtypes can be activated by different PAMPs,which were found on the wall of bacteria. TLR bind ligand on the surface of pathogenic microorganisms, and activate the downstream nuclear factor-κB and MAPK signaling pathways mainly through the MyD88-IRAK-TRAF6-kinase pathway,and then a variety of cytokines, chemokines, co-stimulatory factor and non-specific anti-microbial peptide were released subsequently, induce innate and aquired immune response against the micro-organisms after immune cells maturation and activation to clear that. TLR not only activate the innate immunity,but aslo provide the co-stimulate singal for acquired immunity, so it plays a important role in connecting innate immunity with acquired immunity.Recent studies have confirmed that Propionibacterium acne actives monocytes via TLRs, releases cytokines and induce inflammatory response. This purpose of this research was that the condition of the expresstion of toll-like receptor2(TLR2)in the peripheral blood of patients with acne and the relationship between IL-8,TNF-αand TLR2, and the result eventually indicated that the expression of TLRs in the peripheral blood significantly increased,and has a positively correlated to IL-8,TNF-αand TLR2, all of which given the further proof that TLRs participated the pathogenesis of acne through up-regulated the expression of acne, and induced the production of a large number of inflammatory cytokine,induce inflamation, lead to nflamatory lesions.It is confirmed that TLRs mediated inflammatory injury as a important receptor. A series of experiments proved that Propionibacterium acnes-induced immune response is mainly mediated via TLR2, and TLRs can be found in the lesions, especially around the follicles, has a positively correlated with the duration of disease. After normal primary cultured human monocytes stimulated by different concentrations of propionibacterium acnes that handled by ultrasoni cation, IL-12 and IL-8 were seen a dose-dependent increasing obviously,which could be blocked by anti-TLR2 antibody. Kim confirmed that Propionibacterium acnes activated monocytes through TLR2-dependent pathway, and also found that monocytes in acne lesions expressed TLR2, but not expression of lymphocytes. Mochizuki H aslo found that Propionibacterium acnes activated monocyte,induced to the release of cytokines mediated by TLR2 while the relatively ligand was peptidoglycan on cell wall of propionibacterium acnes. Propionibacterium acnes activated NF-κB signaling pathways TLR2, subsequently activiated innate immune system through PAMPs after binding to PRRs,Firstly, TLR2 recognized peptidoglycan on cell wall of propionibacterium acnes, then formed heterodimers with TLR6,transfered singling and activated downstream effector via TLR/IL—1R, binded TLR receptor located C-terminal domain of adaptor MyD88, Adapter protein MYD88 through the N-terminal death domain connected with the interleukin-1 receptor associated kinase (IRAK) of the N-terminal death domain in the following. The interaction between them lead to phosphorylation and activation,TNF-6 plays a key role in the connection IRAKand IRAK2 to NF-κB belonged to Mitogen-binding protein kinase (MAPK) family, induce kinase activation. NF-κB laterly was activated and transferred to nucleus, triggered immune and inflammatory responses were induced by cytokines,such as IL-8,TNF-α,which were the main reasons caused inflammatory lesions.This research focus on the expresstion of toll-like receptor2(TLR2)in the peripheral blood of patients with Acne and the relationship between IL-8,TNF-a and TLR2, study the pathogenesis in the acne.Methods:Total 30 patients selected during January to June,2009, these who were diagnosed according to clinical dermatology, and consistented with Pillsbury classification involving moderate to severe. All patients with acne did not took any retinoic acid, steroids, immunosuppressive agents within 2 months and did not use topical drugs near 2 weeks.20 normal subjects who have no immune,systemitic, infectious diseases were choosed as control group. There was no significient statistical difference between the control and the patients.5ml peripheral blood was collected after steriling, about 3 ml of which used for flow cytometry, the rest centrifuge for 10 minutes (2500 r/min) under the low temperture, draw the upper set of serum carefully and preserved it in refrigerator at -20℃. The expresstion of TLR2 on the surface of CD14+ cell was observed using multi-color immunofluorescence flow cytometry, the ELISA was used for the detection of the concentation of IL-8、TNF-a.The difference between the paitents and the control were compared using T test,and the value R was caculated using pearson analysis to compare the association between TLR and IL-8、TNF-α.Results:1 The expression of CD14+ cells in peripheral blood of patients with acne:The expression rate and intensity of TLR2 was (50.88±3.14)% and (2.44±0.34),significantly higher than that of normal controls. (P<0.01)2 The serum levels of IL-8 and TNF-αwere significant increase in patients with acne as compared with normal controls.There was significant difference between patients with acne and normal controls (P<0.01). Caculation of the association between TLR and IL-8、TNF-αusing Pearson analysis indicated that the expression of TLR2 was correlated with positively with the concentration of IL-8 and TNF-α. The expression rate of TLR2 was correlated with serum IL-8 and TNF-αlevel,(r1=0.966, r2=0.739), (P<0.01)。Conclusions:The expression of CD14+ cells in peripheral blood of patients with acne is significantly higher than that of normal controls. Caculation of the association between TLR and IL-8、TNF-αusing Pearson analysis indicated that the expression of TLR2 was correlated with positively with the concentration of IL-8 and TNF-α. The TLR2 and the innate immune response mediated by it may play a critical role in the pathogenesis of acne vulgaris, which may be through spurring the excretion of inflammatory factor.更多还原