【作者】 马蕾；

【导师】 高兴华；

【作者基本信息】 中国医科大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 前言特应性皮炎(atopic dermatitis,AD)病因与发病机制尚不清楚,可能是遗传因素、免疫因素、有缺陷的皮肤屏障功能以及环境因素共同作用的结果。神经免疫因素及皮肤屏障功能障碍在AD发病机制中的作用是目前研究的热点。脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)是神经营养因子(neurotrophins,NTs)家族成员之一,除了具有营养神经作用外,也是炎症反应过程中免疫细胞与神经细胞间的重要调节成分。BDNF在AD发病过程中扮演重要的角色。BDNF血清水平与AD患者的疾病活动性、严重性及生活质量相关。BDNF能够诱导AD患者外周血嗜酸性粒细胞趋化、抑制其凋亡;抑制单个核细胞Th1型细胞因子IFN-γ及IL-12的产生,对Th2型细胞因子IL-4、IL-10和IL-13的产生没有影响,进而导致Th1/Th2型细胞因子平衡紊乱、增加抗原特异性IgE的产生。BDNF基因定位于11号染色体短臂11p13,存在多种基因变异和基因多态性,C270T及G196A多态性的研究倍受关注。最近欧洲有少数BDNF基因C270T及G196A多态性与变应性疾病相关性的研究报道,目前尚无AD与BDNF基因多态性在中国人群中的研究报道。角质形成细胞不仅是构成体表的屏障,同时也是一种重要的免疫细胞,可分泌多种细胞因子、表达相应细胞因子受体。源自角质形成细胞的细胞因子可以影响炎症细胞的迁移、角质形成细胞本身的增生和分化,也可增强或减弱其它细胞因子的分泌,并辅助T细胞免疫应答,甚至对全身免疫机能也有重要作用。角质形成细胞表达BDNF受体TrkB,并发生TrkB样免疫反应。BDNF已被证实调控角质形成细胞的增殖反应,维持表皮内环境的稳定;但BDNF如何调节角质形成细胞分泌免疫调节型细胞因子、参与皮肤变应性炎症反应尚需进一步研究。丝聚合蛋白是存在于人表皮角质形成细胞内的一种易溶于水的蛋白,其生成的天然保湿因子(natural moisturizing factor,NMF)对保持皮肤的水合作用及屏障功能至关重要。丝聚合蛋白基因(filaggrin,FLG)位于人类染色体1q21.3区,是表皮分化复合物基因簇的成员之一,与细胞膜形成及表皮终末分化密切相关。FLG基因突变引起的皮肤屏障功能障碍在AD发病中扮演着重要角色,丝聚合蛋白的减少和缺失,可能是引起AD等干燥性皮肤病的主要原因。R501X无义突变与2282del4移码突变是欧洲AD人群FLG基因的主要突变位点;然而,FLG基因突变具有种族及地域差别,在亚洲及非洲AD人群中,并未见R501X及2282del4突变。在日本AD人群中,S2554X无义突变与3321delA移码突变为主要突变位点;但在欧洲AD人群中,未见S2554X与3321delA突变。目前尚无FLG基因突变在中国AD人群的研究报道。本研究采用聚合酶链反应-限制性片段长度多态性(polymerase chainreaction-restriction fragment length polymorphism,PCR-RFLP)结合测序(DNAsequencing)的方法检测BDN F基因C270T、G196A位点多态性以及FLG基因E2422X、Q2417X、S2554X、S2889X、S3296X、R4307X无义突变和7945delA移码突变在中国AD人群的分布,从分子遗传学角度探讨AD的发病机制。同时,我们采用Real-time RT-PCR及ELISA方法检测BDNF对表皮HaCaT细胞分泌免疫调节型细胞因子IL-10、IL-6、IL-12及IFN-γ的影响,以探讨BDNF介导变应性炎症反应的可能机制及BDNF在AD皮损局部细胞因子网络中可能发挥的作用。材料和方法一、研究对象1、病例组181例AD患者,符合Williams诊断标准。男性96例,女性85例,平均年龄12.9±13.7岁。入选患者均处于疾病活动期,采用SCORAD评分确定疾病的严重程度。所有患者均为辽宁籍汉族人,彼此间无血缘关系,于就诊前6个月未使用糖皮质激素及免疫抑制剂。2、正常对照组197例健康志愿者,男性103例,女性94例,平均年龄31.5±9.4岁。已排除有过敏性哮喘、过敏性鼻炎及荨麻疹、湿疹、枯草热等异位性表现者,辽宁籍汉族人,彼此间无血缘关系。以上两组研究对象均已排除精神、神经系统疾患,体重指数正常范围内,签署知情同意书。3、HaCaT角质形成细胞株二、实验材料EDTA、氯仿、异丙醇、无水乙醇、2×Taq Plus Master-Mix聚合酶、琼脂糖、8%非变性聚丙烯酰胺溶液、限制性内切酶HinfⅠ、Ecol 72Ⅰ、NlaⅢ、BfaⅠ、Hpy188Ⅰ、BamHI、DpnⅡ、DdeⅠ、RPMI1640细胞培养液、青链霉素溶液、胰蛋白酶、PBS、DEPC、RealMasterMix。三、实验方法1、应用PCR-RFLP方法对BDNF基因C270T、G196A等位基因进行分型。2、应用Real-time RT-PCR及ELISA方法检测不同浓度BDNF对HaCaT细胞分泌免疫调节型细胞因子IL-10、IL-6、IL-12及IFN-γ的影响。3、应用PCR-RFLP结合测序的方法,对FLG基因E2422X、Q2417X、S2554X、S2889X、S3296X、R4307X无义突变及7945delA移码突变进行检测。四、统计学分析所有统计学分析过程均采用SPSS13.0软件完成。统计方法主要包括x~2检验、单因素方差分析等,P＜0.05即具有统计学意义。Hardy-Weinberg平衡吻合性检验、组间基因型频率及等位基因频率的差异性比较采用x~2检验,并以优势比(odds ratio,OR)及其95%可信区间(confidende interval,CI)表示相对危险度。结果1、特应性皮炎与脑源性神经营养因子基因C270T及G196A多态性相关性分析(1)AD组和正常对照人群BDNF基因C270T和G196A基因型分布符合Hardy-Weinberg平衡定律。(2)BDNF基因C270T位点T等位基因在AD组和正常对照组间分布有显著性差异(OR=3.130,95%CI1.116～8.778)。(3)BDNF基因C270T位点T等位基因在男性AD和正常对照组间分布有显著性差异(OR=4.1 12,95%CI:1.129～14.973)。(4)BDNF基因C270T位点T等位基因在内源性AD和外源性AD组间分布有显著性差异(OR=3.554,95%CI:1.165～10.844)。(5)BDNF基因G196A位点A等位基因在是否伴有一级亲属异位史AD组间分布有显著性差异(OR=1.579,95%CI:1.021～2.442)。2、脑源性神经营养因子对角质形成细胞分泌细胞因子影响的研究(1)不同浓度BDNF作用24小时后,HaCaT细胞IL-10mRNA表达量及培养液上清中IL-10表达水平在0.1ng/mlBDNF组与0ng/mlBDNF组间无明显变化,1ng/mlBDNF组及10ng/mlBDNF组IL-10表达水平与0ng/mlBDNF组及0.1ng/mlBDNF组间差异均具有统计学意义,且表达水平随BDNF浓度增加而升高。(2)不同浓度BDNF作用24h后,0.1ng/mlBDNF组、1ng/mlBDNF组及10ng/mlBDNF组HaCaT细胞IL-12mRNA表达量与0ng/mlBDNF组间均有显著性差异,且随BDNF浓度的增加而减少;培养液上清中IL-12表达水平在0.1ng/mlBDNF组与0ng/mlBDNF组间比较无明显变化,1ng/mlBDNF组及10ng/mlBDNF组IL-12的表达水平与0ng/mlBDNF组及0.1ng/mlBDNF组间差异均具有统计学意义,且表达水平随BDNF浓度增加而降低。3、特应性皮炎与丝聚合蛋白基因突变的相关性研究AD组及正常对照组FLG基因E2422X、Q2417X、S2554X、S2889X、S3296X、R4307X无义突变及7945delA移码突变的突变率均为0。结论1、BDNF基因C270T位点多态性与AD的发生有关,携带T等位基因的男性及内源AD患者发病的危险性均明显增加。2、BDNF基因G196A位点基因型及等位基因在是否伴有一级亲属异位史AD组间分布具有显著性差异,携带A等位基因的伴有一级亲属异位史的AD患者发病的危险性增加。3、HaCaT细胞经不同浓度BDNF作用后,IL-10mRNA的表达量及培养液上清中IL-10表达水平随BDNF作用浓度的增加呈剂量依赖性升高;IL-12mRNA的表达量及培养液上清中IL-12表达水平随BDNF作用浓度的增加呈剂量依赖性降低。BDNF可能通过调节角质形成细胞分泌不同的免疫调节型细胞因子,改变局部细胞因子环境,在AD患者Th1/Th2型细胞因子平衡紊乱中发挥作用。4、中国东北地区FLG基因E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X及7945delA位点基因型保守,均为野生型。FLG基因突变存在明显的种族及地域区别。更多还原

【Abstract】 IntroductionAtopic dermatitis(AD)is a chronic and relapsing inflammatory skin disorder characterized by intense pruritus and eczematous skin lesions.Genetic,environmental, immunologic and pharmacologic factors are implicated in the pathogenesis of AD. Neural immune factors and skin barrier dysfunction are two major hot spots in the study of AD.Brain-derived neurotrophic factor(BDNF)is the second member of the neurotrophin family.Neurotrophins(NTs)have a wide spectrum of functions outside the nervous system.They appear to be important mediators in the interaction between immune and nerve cells in inflammatory,allergic and autoimmune diseases.BDNF plays an important role in the pathogenesis of AD in that BDNF serum levels provide a useful indicator of disease activity,severity and life quality in AD patients.BDNF inhibits the apoptosis and induces the chemotaxis of eosinophils from AD patients. BDNF can also suppress the production of Th1 type cytokines IFN-γand IL-12 by mononuclear cells,but has no effect on the production of Th2 type cytokines IL-4, IL-10 and IL-13.Consequently,the cytokine balance is skewed toward Th2 type and antigen specific IgE production was enhanced.The BDNF gene locates on human chromosome 11p13,consisting of four short 5’exons with separate promoters and one 3’exon encoding the mature BDNF protein.In the BDNF,several variation and polymorphisms have been reported.The most frequent in research studies include C270T and G196A polymorphisms.Recently,a few studies have been reported in the association of BDNF gene C270T and G196A polymorphisms and atopic diseases in European population.However,until now,none of the above polymorphisms has ever been reported in Chinese AD. Keratinocytes not only constitute a barrier surface of the skin,but also function as important immune cells.Keratinocytes secrete a variety of cytokines and express the corresponding cytokine receptors.Keratinocyte-derived cytokines can affect the migration of inflammatory cells and the proliferation and differentiation of keratinocyte itself.Keratinocytes can also enhance or diminish the secretion of other cytokines and participate in T-cell immune response,even to play an important role in whole-body immunity.Keratinocytes express BDNF receptor TrkB and develop TrkB-typed immunological reaction.BDNF has been confirmed to regulate keratinocytes proliferation and maintain environmental stability of epidermis,but how BDNF regulate the secretion of immunomodulatory cytokines by keratinocytes and involve in allergic inflammatory response of skin subject to further study.Filaggrin is a kind of water-soluble protein,located in epidermal keratinocytes. The natural moisturizing factor(NMF)produced by filaggrin is crucial to maintain the hydration function and barrier function of skin.FLG resides on chromosome 1q21 within the epidermal differentiation complex(EDC),which is a cluster of genes and gene families coding for proteins involved in terminal differentiation of the epidermis. Heritable skin barrier defects caused by mutations in filaggrin gene(FLG)play an important role in the pathogenesis of AD.Reduced or absent filaggrin expression may partially explain the skin barrier defects seen in patients with AD.FLG mutations may have regional or ethnic differences.The strong association of FLG R501X and 2282del4 mutations and AD development have been replicated in several European populations,but they were absent in non-European populations,such as those of Asian or African origin.In Japanese populations,the 3321delA and S2554X mutations were associated with AD,whereas the two mutations were not found in European AD patients.However,until now,none of the FLG mutations has ever been reported in Chinese AD.In this study,we investigated C270T and G196A polymorphisms in the BDNF gene and mutations E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X and 7945delA in FLG in Chinese AD population by polymerase chain reaction-restriction fragment length polymorphisms(PCR-RFLP)and DNA sequencing technique,and tried to discuss AD pathogenesis from the molecular genetics angle.We also investigated the effects of BDNF on the production of cytokines by keratinocyte to study the possible function of BDNF in the cytokine network of AD lesions.Materials and MethodsSubjects(1)AD group:181 AD patients,aged 12.9±13.7 year,including 96 males and 85 females were diagnosed in Department of Dermatology,First Hospital of China Medical University,conforms to the Williams diagnosis standard.They were all active AD patients and had no blood relationships.They had not been treated with systemic corticosteroids or other immuno-suppressants within 6 months.(2)Normal control group:197 examples,aged 31.5±9.4 year,including 103 males and 94 females were random selected as volunteers for health examination.All of them had not AD,allergic asthma,allergic rhinitis,atopic keratoconjunctivitis and other allergic symptoms so far.There were no blood relationships between them.Committee on Genetics of China Medical University approved the study,and written informed consent was obtained from all participants.All patients had not psychiatric disorders and their body mass index were within normal range.(3)HaCaT cellsMaterialsEDTA,chloroform,dimethyl carbinol,alcohol,agarose,2×Taq Plus Master-Mix DNA polymerase,8%negative polyacrylamide gel electrophoresis(negative PAGE), restrictive enzyme HinfⅠ、Eco172Ⅰ、NlaⅢ、BfaⅠ、Hpy188Ⅰ、BamHI、DpnⅡ、DdeⅠ、RPMI1640 cell culture fluid,penicilin,streptomycin,trypsinase,PBS,DEPC, RealMasterMix.Methods(1)The C270T and G196A polymorphisms of BDNF were genotyped by PCR-RFLP.(2)The effects of BDNF on the production of cytokines IL-10,IL-6,IL-12 and IFN-γby keratinocytes were analysed by real-time RT-PCR and ELISA.(3)Mutations E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X and 7945delA were detected by PCR-RFLP and DNA sequencing technique. Statistical AnalysisAll the statistical analysis of data were performed using the statistical software SPSS(version 13.0),including Chi-squared test,ANOVA,a two-tailed P＜0.05 was considered to be of statistical significance.To determine whether genotype was in Hardy-Weinberg equilibrium,a x~2 wax performed..The genotype frequency and the allele frequency of these genes were compared by x~2,and odds ratio(OR)and 95% confidence interval(CI)indicate the relative risk.Results1.Association of C270T and G196A polymorphisms in the BDNF gene with atopic dermatitis(1)BDNF gene C270T and G196A genotype distributions of AD group and normal control group conform to the Hardy-Weinberg equilibrium.(2)Significant difference was observed between AD patients and healthy controls in the T allelic frequencies of C270T polymorphism(OR=3.130,95%CI:1.116～8.778).(3)Significant difference was observed between the male AD patients and male healthy controls in the T allelic frequencies of C270T polymorphism(OR=4.112, 95%CI:1.129～14.973).(4)Significant difference was observed between the IAD patients and EAD patients in the T allelic frequencies of C270T polymorphism(OR=3.554,95%CI: 1.165～10.844).(5)Significant difference was observed between AD patients with atopy in 1 st-degree relatives and AD patients without this condition in the A allelic frequencies of G196A polymorphism(OR=1.579,95%CI:1.021～2.442).2.Effects of BDNF on the production of cytokines by keratinocytes(1)The IL-10 mRNA and IL-10 expressions in 0.1ng/ml BDNF irritated HaCaT cells were the same levels as those in control cells,while the expressions of 1ng/ml and 10ng/ml BDNF irritated cells were significantly higher after 24 hr irration and the effect is dose-dependent.(2)The IL-12 mRNA expression in 0.1ng/ml,1ng/ml,and 10ng/ml BDNF irritated HaCaT cells were all lower than that in control cells after 24 hr irration,and the decrease is dose-dependent.The IL-12 level in 0.1ng/ml BDNF irritated supernatant was the same to that of control supernatant,while the levels of other irritated cells were significantly lower after 24 hr irration and the effect is dose-dependent.3.Association of mutations in FLG with atopic dermatitisMutations of E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X and 7945delA in FLG were not detected either in AD group or normal control.Conclusion1.C270T polymorphism in BDNF gene is associated with AD and carrying T allele increases the risk of AD to male AD patients and IAD patients.2.There are significant differences in genotype distribution and allele frequencies for the G196A polymorphism of BDNF gene in AD patients with atopy in 1st-degree relatives in comparison with AD patients without this condition,and carrying A increases the risk of AD to patients with atopy in 1st-degree relatives.3.The IL-10 mRNA and IL-10 expressions increase after BDNF irritation in a dose-dependent way.The IL-12 mRNA and IL-12 expressions decrease after BDNF irritation in a dose-dependent way.BDNF may affect the production of cytokines by keratinocyte,then change cytokine enviroment and play a role in the Th1/Th2 cytokines imbalance.4.FLG E2422X,Q2417X,S2554X,S2889X,S3296X,R4307X and 7945delA are all wide types in AD and normal controls in North East China and FLG mutations have regional or ethnic differences.更多还原