【作者】 韩阳；

【导师】 高兴华；

【作者基本信息】 中国医科大学 ， 皮肤病与性病学， 2018， 博士

【摘要】 背景:马拉色菌（Malassezia）是人体及温血动物皮肤表面的常住菌,也是一种条件致病菌。在皮肤微环境变化、宿主防御功能降低等易感因素条件下,马拉色菌可导致花斑糠疹、马拉色菌毛囊炎等皮肤疾病,也可参与脂溢性皮炎、头皮屑、特应性皮炎及银屑病等疾病的发生。脂溢性皮炎（seborrheic dermatitis,SD）是一种常见的皮肤疾病,临床表现为复发性红斑和油腻性脱屑,伴轻度瘙痒。头皮屑（dandruff,D）多表现为肉眼可见头皮鳞屑异常增多,常被认为是一种发生在头皮部位的轻型脂溢性皮炎。脂溢性皮炎/头皮屑的发病与微生物定植、皮脂水平、免疫、皮肤屏障功能及个体易感性相关。马拉色菌与脂溢性皮炎相关性在对抗真菌药物反应的研究中被证实,研究发现,局部应用抗真菌药物可减少皮损处马拉色菌定植的数量,同时可减轻脂溢性皮炎的症状。马拉色菌可作用于皮肤角质形成细胞及免疫细胞诱导非特异免疫炎症反应的发生,可与角质形成细胞表面的Toll样受体2结合,诱导IL-8等细胞因子分泌。在脂溢性皮炎/头皮屑皮损处定植的马拉色菌主要为球型、限制型和糠秕马拉色菌,其定植密度多高于非皮损区,也有报道为低密度或中密度定植。脂溢性皮炎/头皮屑皮损处中除马拉色菌异常定植外,还存在表皮葡萄球菌定植比例升高。表皮葡萄球菌（Staphylococcus epidermidis,S.epidermidis）是定居于人体皮肤的一种常见的条件致病菌。作为皮肤菌群的一种重要构成菌,其定植比例发生改变可能引起其他菌群定植失衡,导致皮肤疾病的发生。与此同时,部分表皮葡萄球菌还可形成生物被膜样的结构参与致病。近期研究发现,表皮葡萄球菌可产生脂磷壁酸,在多种疾病中发挥抗炎作用,其在马拉色菌相关的皮肤疾病中是否发挥同样的抗炎作用,尚不清楚。由于马拉色菌和表皮葡萄球菌在脂溢性皮炎/头皮屑皮损处同时存在定植比例异常,因此我们猜测马拉色菌与表皮葡萄球菌之间可能存在某种相互作用,参与脂溢性皮炎/头皮屑的发生和发展。前期研究发现糠秕马拉色菌上清液（the supernatant of M.furfur,SMF）对表皮葡萄球菌（ATCC^?12228?）的增殖有促进作用。鉴于以上研究,本研究拟回答以下几个问题:糠秕马拉色菌上清液对表皮葡萄球菌生物膜形成有无影响?糠秕马拉色菌上清液对表皮葡萄球菌蛋白表达有何影响?糠秕马拉色菌上清液通过何种途径影响表葡葡萄球菌增殖及生物膜形成?糠秕马拉色菌诱导角质形成细胞表达何种细胞因子?加入脂磷壁酸后,细胞因子的表达有何变化?为回答上述问题,本研究从菌群相互作用入手,首先采用定量的方法确定糠秕马拉色菌上清液对表皮葡萄球菌增殖的促进作用,并应用结晶紫半定量方法、激光共聚焦显微镜和荧光显微镜观察生物膜的形成,采用蛋白组学方法分析糠秕马拉色菌上清液中的成分以及表皮葡萄球菌受糠秕马拉色菌上清液影响而差异表达的蛋白,寻找糠秕马拉色菌上清液影响表皮葡萄球菌增殖及生物膜形成的机制并确证。应用Real-time PCR、ELISA和Western blot等方法观察葡萄球菌脂磷壁酸对糠秕马拉色菌诱导的细胞因子及NF-κB通路相关蛋白表达的影响,为明确菌群相互作用与脂溢性皮炎/头皮屑相关性奠定基础。材料与方法:1.实验对象:糠秕马拉色菌（ATCC^?14521?）、表皮葡萄球菌（ATCC^?12228?）购于美国ATCC,表皮葡萄球菌（ATCC^?35984?）由复旦大学医学分子病毒学教育部/卫生部重点实验室瞿涤教授惠赠。细胞系选择为人角质形成细胞系（HaCaT）。2.糠秕马拉色菌上清液制备:用牛肉膏蛋白胨氯化钠液体培养基（Beef extract-sodium chloride-peptone,BSCP）将糠秕马拉色菌浓度调整为1×10⁷ colony forming units（CFUs）/mL,于30℃培养15小时后离心、滤除菌体收集培养上清液。3.表皮葡萄球菌增殖测定:每小时检测1次96孔板中细菌的OD值,收集数据至菌体生长曲线达平台期,依据OD_600nm值绘制生长曲线。于37℃培养后12小时,从另一块96孔板各孔中吸取出全部液体放入对应流式管中,冲洗孔壁将冲洗液加入对应的流式管中,用流式细胞仪计数表皮葡萄球菌的数量。4.表皮葡萄球菌（ATCC^?35984?）生物膜检测:采用结晶紫半定量方法观察糠秕马拉色菌上清液对表皮葡萄球菌生物膜形成的影响,依据染色后测定的OD_570nm值进行统计分析。应用激光共聚焦显微镜和荧光显微镜在镜下观察生物膜的形成。5.糠秕马拉色菌上清液蛋白质组学分析及表皮葡萄球菌（ATCC^?12228?）差异蛋白组学分析:采用质谱和生物信息分析方法对糠秕马拉色菌上清液中的蛋白质及受到糠秕马拉色菌上清液影响的表皮葡萄球菌中差异表达的蛋白质进行分析,了解糠秕马拉色菌上清液中的蛋白成分及其对表皮葡萄球菌蛋白质表达的影响。6.平行反应监测法（parallel reaction monitor,PRM）及Real-time PCR检测目标蛋白在蛋白及mRNA水平的表达:采用PRM验证目标蛋白的表达。提取菌体蛋白后,胰酶酶解,液相色谱-质谱联用分析肽段,处理数据,采用峰面积对目标蛋白定量。应用Real-time PCR方法检测目标蛋白mRNA的表达,按照天根RNA提取说明书提取总RNA,用FastQuant RT Kit试剂盒反转录合成cDNA,采用SuperReal PreMix Plus试剂盒进行PCR扩增,采用相对定量法分析结果。7.pH值、NH₄⁺离子、脲酶及脲酶活性检测:应用pH计检测pH值;采用酚次氯酸钠显色法检测NH₄⁺浓度,在640nm波长比色测定,收集数据,分析SMF和BSCP中NH₄⁺离子的浓度;将糠秕马拉色菌接种到脲酶检测培养基中,培养15-72小时,观察培养基是否变色;应用脲酶活性检测试剂盒测定糠秕马拉色菌的脲酶活性,依据测定OD_670nm值,计算脲酶活性。8.透射电镜观察糠秕马拉色菌的形态:将不同脂质条件下培养的糠秕马拉色菌经固定、脱水、包埋、切片后在透射电镜下观察菌体形态。9.Real-time PCR和ELISA检测细胞因子的表达:采用Real-time PCR和ELISA方法检测细胞因子的表达。Real-time PCR操作步骤同前。ELISA检测在加样、洗板、孵育抗体、洗板、显色后用酶标仪检测吸光度。10.Western blot检测NF-κB通路相关蛋白的表达:采用Western blot方法检测细胞P65,p-P65,IκBα及p-IκBα蛋白的表达。用BCA方法测蛋白浓度、跑胶、转模、封闭、孵育抗体、ECL显色拍照及灰度分析。11.统计学分析:应用GraphPad Priam 6软件对数据进行分析及图表绘制,每组样本采用均值±标准误（Mean±SEM）来表示,两组比较采用unpaired t test,多组组间比较采用单因素方差分析。p<0.05时认为差异有统计学意义。结果:1.糠秕马拉色菌上清液对表皮葡萄球菌增殖及生物膜形成的影响:生长曲线显示,糠秕马拉色菌上清液可以促进表皮葡萄球菌（ATCC^?12228?）增殖,且促进作用具有浓度依赖性。流式细胞仪计数发现糠秕马拉色菌上清液原液组和5倍稀释组表皮葡萄球菌数量增多,与对照组相比差异有统计学意义（p<0.05）。结晶紫半定量方法检测生物膜发现糠秕马拉色菌上清液可促进生物膜形成（p<0.05）。激光共聚焦显微镜可见糠秕马拉色菌上清液培养的表皮葡萄球菌（ATCC^?35984?）形成的生物膜结构致密,而对照组形成的生物膜结构较为疏松。2.糠秕马拉色菌上清液中蛋白质分析:共鉴定到102种蛋白质,其中1/3为假定存在的未定性的蛋白质,1/3为基因组构成蛋白,余下1/3为明确了功能的蛋白质,如:蛋白酶、脂酶、磷脂酶等。3.表皮葡萄球菌（ATCC^?12228?）差异蛋白分析:对SMF及BSCP培养后表皮葡萄球菌表达的差异蛋白质进行分析。设定差异倍率为1.30时,共有54个蛋白发生上调,59个蛋白发生下调。差异蛋白中与表皮葡萄球菌致病相关蛋白表达均下调。同时发现,有4个表达下调的蛋白被富集在精氨酸生物合成通路中,这4个蛋白中有3个参与精氨酸分解,精氨酸分解产生的氨可改变酸性环境,对表皮葡萄球菌在酸性环境中存活发挥至关重要的作用。由于本研究发现参与精氨酸分解的相关酶表达下降,因此推测糠秕马拉色菌上清液的pH值高于牛肉膏蛋白胨氯化钠培养基的pH值。4.PRM和Real-time PCR验证差异表达的蛋白:PRM验证目标蛋白表达的趋势与蛋白组学中该蛋白表达趋势一致。应用PCR观察4个精氨酸生物合成通路中的蛋白在mRNA水平的表达,发现其中有3个蛋白在mRNA水平呈现表达下降,与蛋白组学表达趋势一致。5.糠秕马拉色菌通过改变上清pH值影响表皮葡萄球菌（ATCC^?12228?）增殖及表皮葡萄球菌（ATCC^?35984?）生物膜的形成:pH值检测发现SMF的pH值高于BSCP的pH值。单独分析pH值对表皮葡萄球菌增殖及生物膜形成的影响,发现与pH 5.0培养条件相比,pH 6.0可促进表皮葡萄球菌增殖及生物膜形成。将SMF与BSCP的pH值调为一致后,SMF无促进作用。6.糠秕马拉色菌通过脲酶升高pH值:应用脲酶检测培养基发现糠秕马拉色菌可产生脲酶。脲酶可以催化尿素水解为氨,氨可自由溶解于水中形成弱碱性液体升高pH值,并产生NH₄⁺。通过检测NH₄⁺,发现SMF中NH₄⁺浓度高于BSCP。应用脲酶抑制剂与糠秕马拉色菌共培养后提取SMF检测pH值,发现pH值升高较少,因此,我们认为糠秕马拉色菌通过其脲酶升高培养基的pH值。7.脂质条件对糠秕马拉色菌的形态及脲酶活性的影响:无脂质条件下培养的糠秕马拉色菌菌体细胞壁略增厚,透光性下降,菌外壁可见更多丝状纤维,内壁锯齿状结构部分消失,变平滑。部分细胞壁受损,菌体内部细胞器可见结构异形、坏死。脲酶活性检测发现在无脂条件下培养的糠秕马拉色菌脲酶活性升高。8.葡萄球菌脂磷壁酸抑制糠秕马拉色菌诱导的HaCaT细胞炎症因子表达:糠秕马拉色菌与HaCaT细胞共培养后1小时,糠秕马拉色菌可诱导HaCaT细胞IL-6及IL-1βmRNA表达升高。脂磷壁酸和糠秕马拉色菌共同处理组HaCaT细胞IL-6及IL-1βmRNA的表达低于糠秕马拉色菌处理组。ELISA方法检测细胞培养上清中细胞因子,与正常培养的HaCaT细胞上清相比,糠秕马拉色菌促进HaCaT细胞分泌IL-6。脂磷壁酸和糠秕马拉色菌共同处理组上清IL-6分泌低于糠秕马拉色菌处理组。9.葡萄球菌脂磷壁酸通过NF-κB通路抑制糠秕马拉色菌诱导的炎症因子表达:将糠秕马拉色菌与HaCaT细胞共培养30分钟后,糠秕马拉色菌可使HaCaT细胞p-P65表达上调。脂磷壁酸和糠秕马拉色菌共同处理组与糠秕马拉色菌处理组相比,p-P65表达水平较低。结论:1.糠秕马拉色菌上清液对表皮葡萄球菌的增殖和生物膜形成有促进作用。2.糠秕马拉色菌具有脲酶活性,可分解尿素产氨,升高培养环境的pH值。培养环境pH值的改变（约从pH5.0到pH6.0）可为表皮葡萄球菌增殖及生物膜形成提供更为适宜的生长条件。3.在无脂质条件下,糠秕马拉色菌形态发生改变,脲酶活性升高。4.HaCaT细胞在糠秕马拉色菌作用下,IL-6及IL-1βmRNA表达增加且细胞培养上清中IL-6分泌增多。葡萄球菌脂磷壁酸可抑制糠秕马拉色菌的刺激作用,降低IL-6及IL-1β的mRNA表达水平,减少IL-6分泌。糠秕马拉色菌可以促进HaCaT细胞P65蛋白的磷酸化,诱导下游细胞因子表达。脂磷壁酸可抑制P65蛋白磷酸化,进而抑制糠秕马拉色菌诱导的细胞因子表达。更多还原

【Abstract】 Background: Malassezia is a genus of yeasts that residents on the skin surface of human and warm-blooded animals and sometimes acts as an opportunistic pathogen.Under the conditions of predisposing factors,such as lower host defense and changes in skin microenvironment,Malassezia could cause tinea pityriasis and Malassezia folliculitis and be associated with the occurrence of skin disease,including seborrheic dermatitis,dandruff,atopic dermatitis and psoriasis.Seborrheic dermatitis(SD)and dandruff(D)are relapsing skin diseases with erythema and scaling.Dandruff was usually considered as a mild non-inflammatory form of SD.Although the pathogenesis of SD/D were still unclear,various internal factors and external environmental factors had been reported to be related to the occurrence and development of SD/D,such as microorganism colonization,serum status,immune state,skin barrier function and individual susceptibility.It was proved in the research of antifungal drug reactions that the genus of Malassezia was associated with SD/D.Researches demonstrated that partial application of antifungal drugs could reduce Malassezia colonization on the skin lesion and alleviate symptoms of SD/D.Malassezia could interplay with keratinocytes and immune cells to induce a nonspecific immune response.Combination of Toll-like receptor 2(TLR2),Malassezia could promote the production of IL-8 from keratinocytes,which participated in the development of SD.Of all Malassezia species,Malassezia globosa(M.globosa),Malassezia restricta(M.restricta)and Malassezia furfur(M.furfur)are the most commonly isolated species from the lesions of SD/D at significantly high rates in comparison with the normal subjects.Besides the disequilibrated colonization of Malassezia on the lesions,an increasing colonization of Staphylococcus epidermidis(S.epidermidis)were also observed in SD/D.Being the prominent part of skin microbiota,the changes in quantity or proportion of S.epidermidis would lead to a disequilibrium of other flora,which might relate to the occurrence of skin disease.Also,the formation of S.epidermidis biofilm was reported to be involved in the pathogenesis of skin diseases.Recent studies found that lipoteichoic acid(LTA)produced by S.epidermidis played anti-inflammation roles in multiple skin diseases,but it was still uncertain that the same anti-inflammation effect could occur in skin disease induced by Malassezia.However,in SD/D conditions,Malassezia and S.epidermidis both showed disequilibrium on skin lesions.We speculated there might be interactions between these two microorganisms which led to an imbalanced microbiota colonization of the skin.Through our former research,the supernatant of M.furfur(SMF)promoted the growth of S.epidermidis.Based on the previous researches,several questions below would be prepared to investigate: What were the effects of SMF on the biofilm formation of S.epidermidis? What were the effects of SMF on protein expression of S.epidermidis? What would it take for SMF to affect the growth and biofilm formation of S.epidermidis? What kind of cytokines would keratinocytes produce after being stimulated by M.furfur and what was the difference if adding LTA?To answer the questions above,our study started with the interactions between Malassezia and S.epidermidis.The promotion of SMF on growth of S.epidermidis was determined by quantitative method.The semi-quantitative crystal violet staining method and confocal microscopy were used to study the biofilm formation.The protein components of SMF and the differential expression proteins of S.epidermidis influenced by SMF were performed by proteomic analysis.The mechanisms behind growth and biofilm formation of S.epidermidis affected by SMF were explored and verified.Real-time PCR,ELISA and Western blot were used to observe the production of cytokines and the expression of proteins in NF-κB signal pathway induced by Malassezia with LTA.Material and Methods: 1.The experimental objects: M.furfur(ATCC?14521?)and S.epidermidis(ATCC?12228?)were purchased from American Type Culture Collection(ATCC).S.epidermidis(ATCC?35984?)were kindly provided by professor Qu di(Key Laboratory of Medical Molecular Virology of Ministries of Education and Health,Shanghai Medical College of Fudan University,Shanghai,China).Immortalized human keratinocyte,Ha Ca T cell line was used in this study.2.Preparation of SMF: M.furfur was adjusted to 1.0×107 colony forming units (CFUs)/m L with Beef extract-sodium chloride-peptone(BSCP)and was put into 30℃ with shaking overnight(15h).After centrifugation and filtration,the supernatant was collected as SMF for further study.3.S.epidermidis growth measurement: S.epidermidis were measured by testing the bacteria turbidity hourly at 600 nm by microplate reader.The growth curve of each group was mapped according to the optical density(OD)values of bacteria at different hours at 37℃.Another plate was prepared as mentioned above at the same condition for 12 hours.The bacteria suspension was removed to a new tube and the number of the bacteria in each sample was counted by the flow cytometer.4.S.epidermidis(ATCC?35984?)biofilm formation: The semi-quantitative crystal violet staining method and confocal microscopy were used to study the biofilm formation.Absorbance was read at 570 nm in microplate reader.The sterile coverslips were examined by confocal laser scanning microscopy and fluorescence microscope.5.Proteome analysis of M.furfur and S.epidermidis(ATCC?12228?): Mass spectrometry and bioinformatics analysis method were used to resolve proteins in SMF and explore the differentially expressed proteins in S.epidermidis which were influenced by SMF.6.Detection the expression of target proteins by parallel reaction monitoring(PRM)and Real-time PCR: The differently expressed proteins(DEPs)were confirmed by PRM analysis.Proteins in SMF were extracted and then digested by trypsin.Peptide fragment was analyzed by liquid chromatography-mass spectrum(LC-MS)and data were processed by quantitative analysis.The expression of m RNA was detected by Real-time PCR.Total RNA was extracted by RNA prep Pure Bacteria kit.c DNA was synthesized from RNA by Fast Quant RT Kit.RT-Q PCR was performed with Super Real Pre Mix Plus kit.Ct values were calculated by relative quantification.7.Measurement of p H value,NH4+,urease and urease activity: The p H value of SMF and BSCP were measured by p H reagent.We used phenol-sodium hypochlorite method to test the NH4+ level in SMF and BSCP.The OD values at a wavelength of 640 nm were used to calculate the urea level.The urease of M.furfur was detected by urease identification medium.The urease activity assay kit was used to test the urease activity of M.furfur in different conditions.The absorbance was measured at a wavelength at 670 nm and the OD values were proportionated to the urease activity.8.Morphological observation by Transmission electron microscopy(TEM): M.furfur was fixed,dehydrated,embedded and sectioned.The sections were stained and then viewed by transmission electron microscopy.9.Measurements of cytokines by Real-time PCR and ELISA: Real-time PCR and ELISA were used to evaluate the effects of Malassezia on the production of cytokines.Operating steps of PCR were the same as above.The procedures of ELISA included adding samples,washing plates,incubating antibodies,washing plates and determining the OD values.10.Analyze the expression of NF-κB pathway related proteins by western blot: The expression of P65,p-P65,IκBα and p-IκBα were detected by western blot.Total protein was extracted,and the concentration was measured with BCA method.Operation sequence included running the gel,transfering patterns,sealing and incubating antibodies.The bands were visualized with ECL Western Blotting Substrate.11.Statistical analysis: All experiments were repeated at least three times and the results were presented as mean±SEM.The figures were generated using Graph Pad 6.0 software.For experiments with only two groups,unpaired t test was performed for comparison.For more than two groups of experiments,the differences between each group were analyzed by one-way analysis of variance(ANOVA).The results were considered statistically significant only if p<0.05.Results: 1.Effects of SMF on the growth and the biofilm formation of S.epidermidis.The growth of S.epidermidis was promoted by SMF compared with the BSCP control group.The promoting effect of SMF showed a concentration-dependent manner.Similar results were obtained when counting bacteria by flow cytometry.Compared with the BSCP group,undiluted SMF group and the 5-fold diluted SMF showed higher promoting effect(p<0.05).The biofilm formation of S.epidermidis was increased after the treatment of SMF.The confocal laser scanning microscopy images confirmed the promoting effect of SMF on biofilm formation.Confocal microscopy detected a dense membrane like biofilm structure in SMF group,while the control group showed a loose biofilm structure.2.Proteins of SMF: Protein analysis of SMF detected 102 proteins.Among these,one third were uncharacterized proteins presumed to be existed,one third were genomic scaffold protein and the rest were proteins with specified function,such as protease,lipase,phospholipase and so on.3.Effects of SMF on proteome of S.epidermidis(ATCC?12228?): proteomic analysis showed that 113 proteins with 54 upregulated and 59 downregulated were significantly affected by SMF with the fold change cut-off being 1.3.Differentially expressed proteins involved in virulence were downregulated.Arginine biosynthesis pathway was enriched on proteome analysis.We detected four proteins involved in arginine biosynthesis.Three of them were major enzymes involved in catalyze Arginine to ornithine,ammonia and carbon dioxide.Ammonia played a vital role in the survival of S.epidermidis under skin acid stress.The expression of related enzymes participated in decomposition of arginine decreased in our study,which indicated that the p H value of SMF was higher than that of BSCP.4.Verification of differently expressed proteins by PRM and Real-time PCR: The expression trends of target proteins verified by PRM were in accordance with those in proteomics.The m RNA expression of 4 proteins in arginine biosynthetic pathway was verified by PCR,and 3 of them presented lower trend,which showed the same trend with results of proteomics.5.The elevated p H value in SMF promoted the the growth and the biofilm formation of S.epidermidis: The p H level of SMF was higher than BSCP.We compared the growth of S.epidermidis and biofilm formation in the absence of SMF at p H5 and p H6 value and found the growth of S.epidermidis was promoted at p H6 value.Adjusting the p H value of BSCP to the same as SMF,we observed no difference in the growth S.epidermidis.6.Urease increased p H value in SMF: Urease produced by M.furfur was detected by urease identification medium.Urease catalyzed hydrolysis of urea into ammonia and led to the increase of p H value in the culture medium.We co-cultured AHA with M.furfur and then extracted SMF,of which the p H value only showed minor increase.Therefore,it was believed that M.furfur increased p H value by urease.7.The effect of lipid conditions on the morphology and urease activity of M.furfur: Cultured in lipid-free condition,the cell wall of M.furfur was slightly thickened and decreased in permeability.More filaments were seen in the outer wall,while the jagged structure of the inner wall disappeared and became smoothed.Part of the cell walls were damaged.Heteromorphism and necrosis were seen in the organelles.Increased urease activity of M.furfur was detected after cultured in lipid-free condition(m Dixon without lipid components)compared with the normal culture medium in m Dixon,suggesting the urease activity of M.furfur was increased without lipid.8.Staphylococcal LTA inhibited the production of cytokines in Ha Ca T cells induced by M.furfur: M.furfur induced IL-6 and IL-1β m RNA expression in Ha Ca T cells after co-cultured for 1h.LTA inhibited the m RNA expression of IL-6 and IL-1β induced by M.furfur.ELISA measurements observed the similar trend of IL-6 in cell cultural supernatant.9.Staphylococcal LTA inhibited the production of cytokines through NF-κB signal pathway: Co-culture with M.furfur for 30 min,the expression of p-P65 in Ha Ca T cells was increased.While p-P65 expression was decreased after pretreatment with LTA.Conclusion: 1.SMF promoted the growth and biofilm formation of S.epidermidis.2.Urease activity existed in M.furfur,which could catalyze hydrolysis of urea into ammonia and increase the p H value in the culture medium.The changing p H values of cultivation environment(approximately from p H5.0 to p H6.0)provided favorable condition for the growth and biofilm formation of S.epidermidis.The increasing quantity of S.epidermidis might cause disequilibrium in skin flora.While the increasing biofilm formation of S.epidermidis might obstruct sweat gland and sebaceous gland,which took part in pathogenesis of skin disease.3.The morphology of M.furfur made a difference and urease activity increased after cultivation in lipid-free condition.4.Induced by M.furfur,the m RNA expression of IL-6 and IL-1β in Ha Ca T cells was elevated and secretion of IL-6 in cell cultured supernatant was increased.While Staphylococcal LTA could inhibit the production of cytokines induced by M.furfur.M.furfur could promote phosphorylation of P65 protein in Ha Ca T cells and then induce the expression of downstream cytokines.LTA could inhibit the phosphorylation of P65 protein,thus inhibiting the production of cytokines induced by M.furfur.更多还原