γ-分泌酶在反常性痤疮皮损中的表达及TNFA-308基因多态性与寻常性痤疮相关性的meta分析

【作者】 仓田；

【导师】 王宝玺；

【作者基本信息】 北京协和医学院 ， 皮肤性病学， 2013， 博士

【摘要】 研究背景：反常性痤疮(Acne inversa, AI)又称化脓性汗腺炎、毛囊闭锁三联征,是一组以反复发生皮肤脓肿、窦道及瘢痕形成为特征的慢性化脓性皮肤毛囊炎症。皮损常发生在有大汗腺分布的皮肤皱褶部位,如腋窝、乳腺褶、腹部褶皱、腹股沟、臀部及颈项部。皮肤损害早期的特征性表现为多孔的黑头粉刺,而后形成脓肿、窦道、瘘管、皮肤瘢痕挛缩,少数患者可继发鳞状细胞癌。常于青春期后起病,可呈家族性或散发,家族遗传模式为常染色体显性遗传。反常性痤疮的发病机制目前尚未阐明,可能是由遗传因素、免疫因素、抽烟、肥胖、雄激素等多种因素共同作用的结果。γ-分泌酶是一种由四个亚基组成的蛋白酶复合体,可切割30余种Ⅰ型跨膜蛋白,包括淀粉样蛋白前体蛋白、Notch受体、CD44、N-钙粘蛋白和E-钙粘蛋白。γ-分泌酶的四个亚基分别为早老素(presenilin, PS)、早老素增强子-2(presenilin enhancer-2, PEN-2)、纳卡斯楚因蛋白(nicastrin, NCT)和前咽缺陷蛋白-1(anterior-pharynx-defective-1, APH-1)。分别由PS-1/PS-2、PSEN-2、NCT和APH-1a/APH-1b编码。PS可由PS-1或PS-2编码,APH-1可由APH-1a或APH-1b编码,但是每个γ-分泌酶中只可能出现两者之中的一个。所以至少有6种不同的Y-分泌酶变种存在,可能是组织或细胞特异的。本课题组前期研究了6个中国人AI家系,发现在1个家族中出现了PS-1基因的移码突变,在2个家族中出现了PSEN-2的移码突变,在3个家族中分别出现了NCT基因的无义突变、移码和剪接突变。此发现使AI发病机制和γ-分泌酶的关系成为研究热点,但是γ-分泌酶在皮肤中的表达尚未见研究报道。目的：(1)检测PS-1, PS-2, PSEN-2, NCT和APH-1a在AI患者皮损组织和正常皮肤组织中的差异表达。(2)定位PS-1, PS-2, PSEN-2和NCT在AI患者皮损组织和正常皮肤组织中的差异表达部位。方法：(1)提取正常皮肤组织和AI皮损组织总蛋白,用蛋白质免疫印迹法(Westernbloting, WB)检测PS-1, PS-2, PSEN-2, NCT和APH-1α的表达。(2)用免疫组化法(Immunohistochemisty, IHC)定位PS-1,PS-2,PSEN-2和NCT这4种蛋白在AI组织中的表达。结果：(1)PS-2在患者的汗腺和表皮部位显著低表达(汗腺：t=-11.145,p=0.000<0.05；表皮：t=-3.051, p=0.038<0.05), NCT在患者的毛囊和表皮部位低表达(毛囊：t=2.594,p=0.081<0.1；表皮t=-2.376,p=0.098<0.1)。(2)NCT在2名患者(HS3, HS4)和个别对照组正常皮肤中(IVD5)都观察到不表达。(3)APH-1α在2例对照组正常皮肤中(IVD4、IVD5)不表达。结论：(1)PS-2在汗腺部位和表皮的低表达可能与AI发病相关。NCT在毛囊部位和表皮的低表达可能与AI发病有关,NCT蛋白不表达可能并不能独立致病。APH-1a蛋白的不表达可能并不能独立致病。(2)γ-分泌酶在AI发病机制中的作用可能同时影响两个部位,即毛囊和汗腺。研究背景：寻常性痤疮为慢性炎症性毛囊皮脂腺疾病,是皮肤科最常见的疾病之一。寻常性痤疮是多因素致病的共同结果,主要包括性激素水平、皮脂大量分泌、痤疮丙酸杆菌增殖,毛囊皮脂腺导管的角化异常及炎症。近期有关发病机制的文献报告较多,有报道模式识别受体被活化之后,角质形成细胞的细胞质中开始一个快速的信号级联放大反应,导致下游靶基因的增强表达,这些基因编码的细胞因子主要包括肿瘤坏死因子(Tumour necrosis factor,TNF)和IL-1。编码TNF-α的TNFA基因位于6号染色体短臂(6p21.3)主要组织相容性Ⅲ类区域,此区域以丰富的基因多态性为特点。TNFA启动子存在不同类型的基因多态性,包括单核苷酸多态性(Single nucleotide polymorphism, SNPs)和微随体。细胞因子TNF-α是多种生物学进程的关键分子,他的错误调控可损害宿主,启动子区的SNPs可以在基因表达的等位基因调节中发挥作用,并且常被认为是炎症性疾病、感染性疾病以及某些肿瘤性疾病的保护性因素或易感性因素。目的：近年来关于基因多态性与寻常性痤疮易感性的研究文献增多,得到不同结果,但尚无相关meta分析研究。为了更深入的理解基因多态性与寻常性痤疮的相关性,我们进行了本次研究。迄今为止有6篇文献报道TNFA启动子区域SNPs在寻常性痤疮发病机制中的作用。TNFA-308G>A基因多态性在6个报道中都有研究,但得出了不同的结论。此meta分析研究’INFA-308基因多态性是否和寻常性痤疮具有相关性。方法：(1)充分检索寻常性痤疮易感性和基因多态性文献,筛选出符合纳入条件的病例对照研究。(2)对TNFA-308G>A基因多态性和寻常性痤疮相关性研究进行meta分析并按人种进行亚组分析。结果：6篇TNFa-308G>A基因多态性和寻常性痤疮相关性的研究中共801例患者和909例对照被纳入分析。在总体人口中未发现TNFA-308G>A的等位基因A和寻常性痤疮有相关性。但是在亚组分析中,蒙古人种以及高加索和蒙古混血人种(土耳其人)的等位基因A与G对比研究中发现次等位基因A与寻常性痤疮的发病显著相关(蒙古人种OR=2.61,95%CI=1.57-4.33,高加索和蒙古混血人种OR=4.14,95%CI=2.25-7.61)。蒙古人种以及高加索和蒙古混血人种(土耳其人)的TNFA-308A主导基因型(AA+GAvsGG)对比发现主导基因型与寻常性痤疮的发病显著相关(蒙古人种OR=2.55,95%CI=1.44-4.51,高加索和蒙古混血人种OR=4.70,95%CI=2.43-9.09)。在亚组分型高加索人中未发现TNFA-308A等位基因和寻常性痤疮的相关性。结论：(1)中国人和土耳其人中TNFA-308G>A的次等位基因A可能和寻常性痤疮易感性相关,但高加索人次等位基因A和寻常性痤疮易感性无相关。(2)中国人属蒙古人种,土耳其人属高加索人种地中海类型,混有蒙古人种成分。提示TNFA-308G>A的次等位基因A在蒙古人种中和寻常性痤疮的发病有相关性。更多还原

【Abstract】 BackgroundsAcne inversa (AI, Hidradenitis suppurativa,Follicular occlusion triad) is a chronic inflammatory skin disease that presents with comedones, painful nodules, abscesses, and sinus tracts in apocrine gland-bearing areas. Longstanding disease can result in fibrosis, dermal contractures, significant scarring, formation of fistulae, and rarely malignant transformation to squamous cell carcinoma.AI affects most commonly the axillae, submammary folds, abdominal fold, groin, buttocks, and neck. AI typically occurs after puberty and can be either familial or sporadic. Familial AI usually shows an autosomal dominant inheritance pattern.The pathogenesis is obscure, but suggested contributory factors include a genetic predisposition, immunologic abnormality,smoking,obesity and androgens.y-Secretase is an intramembranous protease complex capable of cleaving in excess of30type-I transmembrane proteins including amyloid precursor protein, Notch receptors, CD44,N-cadherin, and E-cadherin. The complex is composed of four hydrophobic proteins, presenilin, presenilin enhancer-2, nicastrin,and anterior pharynx defective-1, encoded by PS-1/PS-2, PSEN-2,NCT, and APH-la/APH-lb, respectively. Presenilin can be encoded by PS-1or PS-2and anterior pharynx defective-1can be encoded by APH-1A or APH-1B, but only one of each can be present in any one y-secretase complex, and thus at least six differenty-secretase variants exist and these may be tissue-or cell-specific.In our previous study,we collected samples from six Han Chinese families with features of AI, and identified a frameshift mutation in PS-1in one family, a frameshift mutation in PSEN-2in two families, nonsense mutation,frameshift mutation and splicing mutation in NCT.These mutations highlighted y-secretase to have an integral role in AI. Basic questions, such as the expression of the y-secretase subunits in human skin, need to be addressed.Objectives(1) To verify the differential expressions of PS-1, PS-2, PSEN-2, NCT and APH-la between AI lesional skin tissues and normal skin tissues.(2) To localize the sites of differential expressions of PS-1, PS-2, PSEN-2and NCT between AI lesional skin tissues and normal skin tissues. Methods(1) The total protein lysates were obtained from AI lesional skin tissues and normal skin tissues and were analysed by Western bloting to detect the expressions of PS-1, PS-2, PSEN-2, NCT and APH-la.(2) Immunohistochemistry (IHC) was used to localize the sites of differential expressions of PS-1, PS-2, PSEN-2and NCT between AI lesional skin tissues and normal skin tissues.Results(1) Low expression of PS-2protein was found in sweat glands and epidermis of AI lesional skin tissues(sweat glands:t=-11.145, p=0.000<0.05; epidermis:t=-3.051, p=0.038<0.05).Expreesion of NCT protein was reduced in hair follicle and epidermis of AI lesional skin tissues (hair follicle:t=2.594, p=0.08<0.1; epidermis:t=-2.376, p=0.098<0.1)(2) No expression of NCT protein was observed in two AI lesional skin tissues (HS3, HS4) and one normal skin tissues (FVD5).(3)No expression of APH-la protein was noticed in two normal skin tissue (IVD4,IVD5)Conclusions(1) Low expression of PS-2in sweat glands and epidermis is likely to play a role in the pathogenesis of AI. Likewise, low expression of NCT in hair follicle and epidermis may also play a role in the pathogenesis of AI. In contrast, no expression of NCT, as well as APH-la, might not act independently in causing the disease.(2)γ-secretase appears to be able to subtly affect hair follicle and sweat glands in the pathogenesis of AI. BackgroundsAcne vulgaris is a chronic disorder of the pilosebaceous apparatus.The cause of the disease is unknown, but suggested contributory factors include hormone, an increase sebum secretion, Propionibacterium acnes, abnormal keratinocyte function and inflammation.Upon the activation of pattern recognition receptors, a rapid signaling cascade is initiated in the cytoplasm of the keratinocytes, leading to the increased expression of downstream target genes, which include genes encoding the cytokines TNF-α and interleukin-1(IL-1). The TNFA gene encoding the TNF-α cytokine is located on the short arm of chromosome6(6p21.3) in the major histocompatibility class Ⅲ region, which is characteristic of a high degree of genetic polymorphism. Different types of polymorphisms exist in the TNFA promoter, including SNPs and microsatellites, whereas the coding region is highly conserved The cytokine TNF-α is a key molecule in various biologic processes,and its misregulation may have deleterious effects on the host organism. The SNPs in the promoter region can play a role in the allele-specific regulation of gene expression and are often reported to act as protective or disease-predisposing factors in the development of inflammatory and infectious diseases and certain types of cancers.ObjectiveFor the past few years, gene polymorphisms of acne vulgaris were frequently investigated. However, since the currently published studies only referred to a modest sample size and unified ethnicity, which might not achieve a reliable conclusion, proposing that a meta-analysis is needed to pool and analyze the current data. To date, there have been6articles concerning the role of TNFA promoter SNPs in the pathogenesis of acne vulgaris. The TNFA-308G>A polymorphism was common in all of them, but the conclusions were different. The aim of this meta-analysis is to make precise estimate regarding associations between TNFA-308polymorphisms and acne vulgaris.MethodsBased on an extensive literature search to identify all case-control studies of relevant subjects, we conducted a meta-analysis to assess the association between TNFA-308G/A polymorphisms and susceptibility to acne vulgaris. We also performed subgroup analyses targeting ethnicity.ResultsAmong studies investigating such association6were identified to be eligible, including801patients and909controls. No significant association was observed in all genotypes in overall populations, but analysis with stratification by ethnicity indicated that the TNFA-308A allele in comparison with the G allele, was associated with an increased risk of acne vulgaris in Caucasian and Mongoloid Mixed (Trukese) individuals (OR=4.14,95%CI=2.25-7.61) and Mongoloid individuals (OR=2.61,95%CI=1.57-4.33). Similar results were obtained when the AA+AG genotype was compared with the GG genotype (OR=4.70,95%CI=2.43-9.09) in Caucasian and Mongoloid Mixed (Trukese) individuals and Mongoloid individuals (OR=2.55,95%CI=1.44-4.51) except Caucasian individuals.ConclusionThe results of this meta-analysis indicate that the TNFA-308A allele may be an important risk factor for acne vulgaris in Caucasian and Mongoloid Mixed (Trukese) individuals and Mongoloid individuals, but it is not likely to confer susceptibility to acne vulgaris in Caucasian individuals.更多还原