【作者】 苏玉文；

【导师】 陆前进；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2010， 博士

【摘要】 银屑病是一种常见的慢性复发性炎症性皮肤病,伴有表皮过度增生和血管增殖。寻常型银屑病是银屑病中最常见的一种类型。银屑病的发病机制复杂,确切的病因及机制尚不清楚。银屑病患者病情常反复发作,大多数患者呈现冬重夏轻的发病规律；最易促发或加重银屑病的因素包括感染、精神紧张和应激事件、外伤、手术、妊娠、吸烟和某些药物作用等。种种迹象表明,环境因素在银屑病的发生、发展和转归中扮演着重要的角色。目前认为,银屑病是具有遗传素质的个体在内外环境因素的共同作用下,由T细胞介导的自身免疫性疾病。已有研究表明,表观遗传调控机制在系统性红斑狼疮等自身免疫性疾病的发生、发展中起重要作用。然而,表观遗传修饰在银屑病发生、发展中的作用和机制目前尚不十分清楚。表观遗传学是研究在DNA序列没有发生改变的情况下,基因异常表达并将这种改变遗传给后代的学科。表观遗传调控机制主要包括DNA甲基化、组蛋白修饰以及microRNA调控等。DNA甲基化是被最早发现并研究最多的表观遗传调控机制之一,广泛存在于细菌、植物和哺乳动物中,是DNA的一种天然修饰方式。DNA甲基化是指在DNA甲基转移酶(DNA methyltransferases, DNMTs)的催化作用下,以S-腺苷甲硫氨酸(S-adenosylmethione, SAM)作为甲基供体,使甲基(-CH3)转移至胞嘧啶5位上,形成5-甲基胞嘧啶(5mC)的生物化学过程。在整个基因组中,CpG岛通常位于基因启动子区域(promoter region);在正常情况下,CpG岛是以非甲基化形式存在于基因的启动子内。DNA甲基化修饰没有改变基因的序列,但是却对基因的表达起到调控作用。一般来说,DNA甲基化可以沉默某些基因的表达,而低甲基化则能够诱导某些基因的表达。到目前为止,已经发现大量的肿瘤抑制基因(tumor suppressor genes)的失活与该基因的启动子区域过度甲基化有直接联系。相反,低甲基化可以导致一些在正常情况下受到抑制的基因如癌基因大量表达,从而导致癌症的发生。组蛋白修饰是另一种重要的表观遗传调控机制。组蛋白是与真核生物DNA结合存在的碱性蛋白质的总称,包含H1、H2A、H2B、H3、H4等5种成分。除H1外,其余4种组蛋白均分别以二聚体(共八聚体)相结合,形成核小体核心；而DNA则缠绕在核小体的核心上。组蛋白翻译结束后,其氨基N末端可发生多种共价修饰,如乙酰化、甲基化、磷酸化、泛素化及ADP核糖基化等,这些修饰状态可以相互联合或被其他蛋白酶或复合体等识别、结合而发挥作用,进而为促进或抑制基因表达的染色质相关蛋白提供结合位点。在上述组蛋白修饰中,研究较多的是组蛋白乙酰化与甲基化。组蛋白N末端富含赖氨酸,在生理状态下携带正电荷,可以与携带负电荷的DNA结合形成致密、高度折叠的核小体。组蛋白乙酰化修饰能够中和正电荷,减弱其与DNA的相互作用,从而导致一个“开放”的染色体结构,利于转录调节因子与DNA的结合。组蛋白乙酰化与基因活化以及DNA复制相关,组蛋白去乙酰化和基因的失活相关。乙酰化转移酶(Histone acetyltransferases, HATs)可以将乙酰基转移至组蛋白H3、H4的N末端,而去乙酰化酶(Histone deacetylases, HDACs)的作用恰恰相反。由此可见,不同位置的修饰需要特定的酶来完成。除了组蛋白乙酰化以外,组蛋白也可以发生甲基化修饰,并且不同位点的组蛋白甲基化修饰对基因的转录具有不同的影响,可以促进基因转录或者抑制基因转录。MicroRNA是近年来在真核生物中新发现的一类参与基因转录后水平调控的非编码小分子RNA。它是一类内源性的、长度约为21-25个核苷酸的小分子,通过与目的基因靶mRNA的3’-UTR碱基序列的不完全或者完全配对,从而导致目的基因翻译的抑制或者基因表达沉默。研究表明,microRNA在生长、发育、细胞增殖和凋亡、脂类代谢及肿瘤形成等许多生理和病理过程中发挥重要作用。MicroRNA普遍存在于多细胞生物中,而且数量十分可观,约占整个基因组基因总数的2%左右。MicroRNA芯片是高通量检测microRNA表达情况的最佳选择,它可以在短时间内同时鉴定所有已知的microRNA的表达。为了探讨DNA甲基化、组蛋白乙酰化/甲基化和microRNA表达在寻常型银屑病发病中的作用及机制,本课题研究了寻常型银屑病患者外周血单个核细胞(Peripheral blood mononuclear cells, PBMCs)及皮损组织中基因组DNA整体甲基化水平、特异性基因甲基化状态以及DNA甲基化修饰酶DNMTs和甲基化结合蛋白MBDs (Methyl-CpG-binding domain protein, MBD) mRNA表达水平；同时检测了组蛋白H3/H4乙酰化水平、H3-K4/H3-K27甲基化水平及组蛋白乙酰化酶HATs (Histone acetylases, HATs)和去乙酰化酶HDACs (Histone deacetylases, HDACs) mRNA表达水平；检测寻常型银屑病患者PBMCs中microRNA表达谱,并对芯片结果进行验证。通过对寻常型银屑病患者PBMCs和皮损组织表观遗传修饰的研究,探讨表观遗传调控机制是否参与寻常型银屑病的发病,进一步全面揭示银屑病的发病机制。目的研究寻常型银屑病患者PBMCs和皮损组织DNA甲基化状态及甲基化相关调控基因的表达,从而探讨DNA甲基化在寻常型银屑病发病中的作用和机制。方法密度梯度离心法分离30例寻常型银屑病患者和20例正常对照者PBMCs,应用整体甲基化定量试剂盒检测寻常型银屑病患者和正常对照者PBMCs基因组DNA整体甲基化水平；实时荧光定量聚合酶链反应(Real-time PCR)检测DNMTs和MBDs的mRNA表达水平；收集30例寻常型银屑病患者皮损组织和15例正常人皮肤组织,采用免疫组化法检测皮损组织中5-甲基胞嘧啶表达水平。此外,采用Real-time PCR法和甲基化特异性PCR(MSP)法分别检测皮肤组织中p14ARF mRNA的表达水平和甲基化状态。结果与正常对照组相比,寻常型银屑病患者PBMCs和皮损组织中基因组DNA整体甲基化水平增高(p=0.011和p=0.007),皮损组织免疫组化评分与银屑病皮损面积和严重程度评分(Psoriasis Area and Severity Index, PASI)具有显著正相关性(r=0.868,p=0.000)；寻常型银屑病患者PBMCs中DNMT1 mRNA表达水平明显增高(p=0.011),MBD2和MeCP2 mRNA表达水平明显降低(p=0.043和p=0.000)。此外,和正常对照组相比,寻常型银屑病患者皮损组织中抑癌基因mRNA表达水平明显降低(p=0.006),通过MSP法证实抑癌基因p14ARF启动子处于高甲基化状态(p=0.036)。结论寻常型银屑病患者PBMCs和皮损组织整体基因组DNA及抑癌基因p14ARF启动子处于高甲基化状态,且甲基化相关调控基因表达异常。目的研究寻常型银屑病患者PBMCs中组蛋白乙酰化/甲基化水平以及组蛋白修饰酶表达水平。方法密度梯度离心法分离30例寻常型银屑病患者和20例正常对照者PBMCs,应用组蛋白乙酰化/甲基化水平检测试剂盒测定组蛋白H3/H4乙酰化水平、H3-K4/H3-K27甲基化水平；Real-time PCR法检测组蛋白乙酰化酶(HATs、HDACs) mRNA表达水平。结果和正常对照组相比,寻常型银屑病患者PBMCs中组蛋白H4乙酰化水平显著降低(p=0.005)；寻常型银屑病患者组中组蛋白H3乙酰化水平、H3-K4和H3-K27甲基化水平均低于正常对照组,但无统计学差异(p>0.05)；寻常型银屑病患者PBMCs中HDAC1 mRNA表达水平显著升高(p=0.005),而P300,CBP和SIRT1 mRNA表达水平显著降低(p=0.024,p=0.006,p=0.002)。结论寻常型银屑病患者PBMCs中组蛋白乙酰化修饰异常。目的研究寻常型银屑病PBMCs中microRNA表达变化。方法密度梯度离心法分离9例寻常型银屑病患者和9例正常对照者PBMCs,用TRIzol试剂提取总RNA及microRNA,应用Affymetrix microRNA基因芯片技术检测PBMCs中异常表达的microRNA,并应用Real-time PCR法对30例寻常型银屑病患者和20例健康对照中进行芯片结果验证。结果Affymetrix microRNA基因芯片结果显示：和正常对照组相比较,miR-210,miR-584,miR193b和miR-501-3p符合差异表达microRNA筛选标准,在寻常型银屑病患者组PBMCs中均高表达(p<0.01)；通过Real-time PCR法验证了上述结果。此外,Real-timePCR结果还显示miR-203在寻常型银屑病患者组PBMCs中表达较正常对照组升高(p<0.01)。结论寻常型银屑病患者组PBMCs中microRNA表达异常。更多还原

【Abstract】 Background Psoriasis vulgaris, a common, chronic and relapsing inflammatory skin disease, is the commonest subtype of psoriasis clinically associated with epidermal hyperplasia and vascular proliferation. The etiology and pathogenesis of psoriasis are still unclear. Patients with psoriasis usually have a recurring manifestation, characterized by aggravation in winter and remission in summer. Factors promoting or aggravating the disease include infection, mental tension and stress events, trauma, surgery, pregnancy, smoking and certain drugs. More and more evidence indicates that environmental factors play significant roles in the occurrence and development of psoriasis. Psoriasis vulgaris is a T-cell-mediated autoimmune skin disease associated with epidermal hyperplasia and vascular proliferation, although putative autoantigen remains unknown. Previous studies demonstrated that epigenetic modification plays an important role in the occurrence and development of autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). However, the role and mechanism of epigenetic regulation in the pathogenesis of psoriasis remains unclear.Epigenetics, referring to stable and heritable changes in gene expression independent of alterations in DNA coding sequence, comprises of DNA methylation, histone modifications and microRNAs.DNA methylation, the most characterized epigenetic modification, takes place almost exclusively at the 5’-carbon position of cytosine residues within CpG pairs, which is catalyzed by DNA methyltransferases (DNMTs) with S-adenosyl-methionine (SAM) as the methyl donor. DNA methylation exists in many kinds of organisms such as bacterial, plants and mammalians, which is a modification style in nature. In general, DNA methyaltion is associated with gene siliencing, while DNA hypomethylation is responsible for gene activation. Up to now, DNA hypermethylation in promoter regions have been found to be associated with gene silencing of a great number of tumor suppressor genes, while DNA hypomethylation will lead to over-expression of genes which are suppressed in normal conditions.Histone modifications are another important epigenetic mechanism. Histones are the alkaline proteins binding to eukaryotic DNA, consisting of H1, H2A, H2B, H3 and H4. An octamer is constructed by H2A, H2B, H3 and H4, surrounded by 146 bp of DNA. The post-translational modifications on each histone subtype include acetylation, methylation, phosphorylation, ubiquitination, ADP ribosylation and so on, which can interact each other or with other factors to provide binding sites for transcriptional regulation proteins activiating or inhibiting gene expression. The study on histone modifications focuses on the acetylation and methylation of lysine residues of N-terminal tails. Histone acetylation can neutralize the positive charge in histones and therefore lossens their interaction with DNA backbone with the negatively charge, resulting in chromatin structure more open and accessible for transcriptional factors. In contrast, methylation in different sites of histones may either activates or repress gene expression.In recent years, microRNAs, a class of newly-discovered small RNA, are believed to be involved in post-transcriptional regulation of gene expression in eukaryotic cells. MicroRNAs are genome-encoded 21-23nt RNAs targeting the 3’untranslated region (3’-UTR) of specific messenger RNAs (mRNA) for degradation or translational repression. It has been proved that microRNAs play important roles in growth, development, cell proliferation, apoptosis, lipid metabolism and tumor formation as well as many other physiological and pathological processes. microRNAs exist in the majority of multi-cell organisms and account for 2% among the genome. Microarray is the best choice to detect the expression of microRNA.To investigate the question whether there are any changes in epigenetic modifications in psoriasis, we detected DNA methylation status, histone acetylation/methylation levels and microRNAs expression in patients with psoriasis vulgaris. Firstly, we detected the global genomic DNA methylaiton status in peripheral blood monouclear cells (PBMCs) and skin lesions as well as mRNA expression levels of DNA methyltransferase (DNMTs) and Methylated CpG binding proteins (MBDs) in PBMCs; secondly, we detected mRNA level and promoter methylation status of p14ARF in skin lesions. We also measured the global histone H3/H4 acetylation and H3-K4/H3-K27 methylation levels in PBMCs and mRNA levels of histone acetyltransferases (HATs) and histone deacetylases (HDACs); finally, miRNA expression profiling and real-time PCR were carried out to identify the aberrant microRNA expression in PBMCs from psoriatic patients. Our study will contribute to uncover whether epigenetic modifications are involved in the occurence and development of psoriasis and establish foundations for comprehensive and deep study on the pathogenesis of psoriasis, thereby providing the novel clues for the diagnosis and treatment for psoriasis. Objective To investigate global DNA methylation and the expression of genes that regulate methylation in patients with psoriasis vulgaris.Methods Global DNA methylation level in PBMCs from 30 psoriatic patients and 20 healthy controls was quantified by an ELISA-like reaction using 5-methylcytosine antibody. Skin samples from 30 psoriatic patients and 15 healthy controls were labeled immunohistochemically with mouse monoclonal anti-5-methylcytosine antibody. The expression of enzymes involved in DNA methylation was measured using real-time PCR. The mRNA level and methylation status of the tumor suppressor gene p14ARF was assessed by real-time PCR and methylation-specific PCR, respectively.Results Compared with healthy controls, PBMCs genomic DNA was significantly hypermethylated in patients with psoriasis vulgaris (p=0.011), and DNMT1 mRNA expression was upregulated (p=0.011). Expression levels of MBD2 and MeCP2 mRNA were significantly lowered in PBMCs from psoriatic patients than healthy controls (p=0.043, p=0.000). Anti-5-methylcytosine labeling was increased in psoriatic skin lesions compared to healthy controls (p=0.007), and there was a significantly positive correlation between 5-methylcytosine labeling and Psoriasis Area and Severity Index (PASI) scores (r=0.868, p=0.000). The tumor suppressor gene p14ARF exhibited decreased mRNA expression (p=0.006) and increased promoter methylation in psoriatic skin lesions compared to healthy skin tissues (p=0.036).Conclusion Aberrant DNA methylation may play an important role in the pathogenesis of psoriasis vulgaris. Objective To investigate global histone H3/H4 acetylation, H3K4/H3K27 methylation levels and the mRNA expression of enzymes involved in histone modifications in patients with psoriasis vulgaris.Methods Global histone H3/H4 acetylation and H3K4/H3K27 methylation in PBMCs from 30 psoriatic patients and 20 normal controls were assayed using the EpiQuikTM global histone H3/H4 acetylation, and H3K4/H3K27 methylation assay kit, respectively. The mRNA levels of enzymes involved in histone acetylation/methylation were measured by real-time PCR.Results Global histone H4 hypoacetylation was observed in PBMCs from patients with psoriasis vulgaris compared with normal controls (p=0.005). There were no significant differences in H3 acetylation and H3K4/H3K27 methylation levels between psoriatic patients and normal controls (p>0.05). The expression of HDAC1 mRNA was upregulated (p=0.005), while mRNA levels of P300, CBP, SIRT1 were decreased in PBMCs from patients with psoriasis vulgaris compared to healthy controls (p=0.024, p=0.006, p=0.002, respectively).Conclusion Abnormal histone acetylation may contribute to development of psoriasis. Objective To investigate the roles of microRNAs in the pathogenesis of psoriasis vulgaris.Methods PBMCs from 9 patients with psoriasis vulgaris and 9 normal controls were extracted by Ficoll-Hypaque density gradient centrifugation. Total RNA including microRNAs was extracted using TRIzol reagent. MicroRNAs with differential expression were detected by Affymetrix microRNAs chips and the results were confirmed in 30 patients with psoriasis vulgaris and 20 healthy controls by real-time PCR.Results The results from Affymetrix microRNAs chips showed that miR-210, miR-584, miR193b and miR-501-3p were overexpressed in PBMCs from patients with psoriasis compared to normal controls (p<0.01), all of which were confirmed in 30 patients and 20 healthy controls by real-time PCR. In addition, the increased expression of miR-203 was also identified in PBMCs from patients with psorisis vulgaris (p<0.01).Conclusion Aberrant microRNAs expression may play a role in the pathogenesis of psoriasis vulgaris.更多还原