【作者】 罗勇奇；

【导师】 陆前进；

【作者基本信息】 中南大学 ， 皮肤病与性病学， 2009， 博士

【摘要】 研究背景亚急性皮肤型红斑狼疮(subacute cutaneous lupuserythematosus,SCLE)是红斑狼疮(lupus erythematosus,LE)的一个亚型,与系统性红斑狼疮(Systemic lupus erythematosus,SLE)患者相比,大部分SCLE患者病情一般进展较慢或者相对温和,很少出现严重内脏器官受累的情况,如SLE患者经常出现的肾脏受累、心血管受累或中枢神经系统受累等。但是,也有部分SCLE患者的病情在经过多年的慢性发展过程后,会逐渐转变成SLE,并出现与SLE患者一样的系统受累的情况。尽管LE的病因尚不清楚,但近年来,越来越多的研究表明表观遗传机制,特别是T细胞DNA低甲基化在T细胞基因调控及SLE发病中起着十分重要的作用,而T细胞DNA低甲基化是否在SCLE的发生、发展中起作用?目前国内外尚无相关的研究报道。DNA甲基化是重要的表观遗传学机制之一,在胚胎发育、遗传印记、X染色体失活、染色质重塑等方面起重要作用。DNA甲基化是指以S-腺苷甲硫氨酸(SAM)作为甲基供体,将其甲基转移到DNA脱氧胞嘧啶(deocytosine)的第5位碳原子,形成5-甲基胞嘧啶(5-mC)的化学修饰过程,通常发生在二核苷酸CpG中的胞嘧啶。基因启动子和增强子区域CpG岛的甲基化可以使一些转录因子无法与DNA结合而抑制基因的转录。DNA甲基化状态受到DNA甲基转移酶(DNA methyltransferase,DNMT)的调控,人类DNMT家族成员主要包括DNMT1、DNMT3a和DNMT3b。DNMT1为维持型甲基转移酶,即使半甲基化的双链DNA变成完全甲基化的双链DNA,确保体内特异甲基化方式在DNA复制过程中得到传递;而DNMT3a与DNMT3b属于新生型甲基转移酶,即催化非甲基化的DNA成为甲基化的DNA状态。近年来,研究结果表明SLE患者T细胞中DNA甲基转移酶(DNMT)活性仅为正常人的1/3-1/2,DNMT1的表达下降,在SLE患者T细胞的甲基化中起重要作用。甲基化修饰后的DNA对基因表达的调控还需要特异结合于甲基化CpG位点(methyl-CpG,mCpG)的蛋白因子参与,因为这些都含有一同样的甲基CpG结合结构域(methyl CpG bindingdomain,MBD),又被称为MBD蛋白家族,包括MBD1,MBD2,MBD3,MBD4和MeCP2。MBD蛋白通过选择性结合甲基化的DNA,与一些相关蛋白协同影响DNA甲基化、染色质构型和基因转录。有报道SLE患者CD4+T细胞MBD2和MBD4的表达增加,并认为其在T细胞的甲基化中发挥重要的作用。近年来的越来越多的研究结果表明T细胞DNA甲基化异常在SLE发病中起十分重要的作用。用DNA甲基化抑制剂处理鼠或人T细胞后,可以使T细胞发生低甲基化,诱发狼疮样的自身免疫反应。而SLE病人T细胞亦表现出总体的基因组低甲基化和自身免疫相关基因的低甲基化,这些基因包括CD11a(ITGAL)、CD70(TNFSF7)、CD40L(TNFSF5)和Perforin(PRF1),过度表达这些基因的T细胞具有自身反应性,杀伤自身巨噬细胞或辅助B细胞产生大量的自身抗体,在SLE的发生、发展中起十分重要的作用。SCLE发病的遗传与表观遗传因素目前仍不清楚。为了探讨DNA甲基化异常在SCLE发病中的作用及机制,本课题研究了SCLE患者外周血T细胞基因组DNA总体甲基化水平以及DNMTs mRNA和MBDsmRNA表达水平,同时检测了CD11a、CD70、CD40L和Perforin这些自身免疫密切相关基因的表达水平及其调节序列的甲基化状态,并分析基因调节序列的甲基化水平与其表达水平的关系。第一部分SCLE患者外周血T细胞DNA总体甲基化异常的研究目的研究亚急性皮肤型红斑狼疮患(SCLE)患者T细胞DNA总体甲基化状态以及甲基化相关调控基因的表达,从而探讨SCLE的表观遗传学改变。方法密度梯度离心分离12例SCLE患者和9例正常人的外周血单个核细胞,磁珠分选CD4+和CD8+T细胞,然后用总体甲基化定量试剂盒检测SCLE患者和正常对照CD4+和CD8+T细胞的DNA总体甲基化水平,实时定量聚合酶链反应(real-time RT-PCR)检测DNA甲基转移酶(DNMTs)和甲基结合蛋白(MBDs)的mRNA表达。结果与正常对照组相比,SCLE患者CD4+T细胞基因组DNA总体低甲基化(P=0.002),而CD8+T细胞的基因组DNA总体甲基化水平无明显变化(P=0.231);SCLE患者CD4+T细胞DNMT1和DNMT3a的mRNA水平明显降低(P值分别为p=0.027和p=0.004),MBD1,MBD3和MBD4 mRNA明显增高(P值分别为p＜0.001,p＜0.001和p=0.001);SCLE患者CD8+T细胞MECP2 mRNA和MBD4 mRNA表达水平较正常对照组明显增高(P值均为p=0.001);我们还发现SCLE患者CD4+T细胞DNA总体甲基化水平与DNMT1mRNA表达水平呈正相关(r=0.590,p=0.044)。结论SCLE患者CD4+T细胞基因组DNA低甲基化,且甲基化相关调控基因表达异常。第二部分SCLE患者T细胞自身免疫反应相关基因表达及其调节序列甲基化状态的研究目的研究SCLE患者外周血T细胞CD11a,CD70,CD40L及Perforin表达及其调节序列的甲基化状态。方法密度梯度离心分离12例SCLE患者和9例正常人的外周血单个核细胞,用流式细胞检测PBMC中CD11a,CD70,CD40L及Perforin蛋白在CD4+和CD8+T细胞中的表达水平;磁珠分选CD4+和CD8+T细胞,实时定量聚合酶链反应(real-time RT-PCR)检测CD11a,CD70,CD40L及Perforin的mRNA表达水平;western-blot检测Perforin的蛋白表达水平;亚硫酸氢钠测序检测表达有差异的蛋白的基因调节序列的甲基化状态。结果与正常对照组相比,SCLE患者CD4+T细胞CD70和Perforin表达增高,且其基因调节序列呈明显的低甲基化。结论SCLE患者CD4+T细胞中CD70和Perforin的过度表达与其基因调节序列的低甲基化相关。更多还原

【Abstract】 Background Subacute cutaneous lupus erythematosus（SCLE） is a subtype of lupus erythematosus（LE）.Most patients with SCLE have a chronic or relapsing but benign condition,with few of the serious manifestations associated with systemic lupus erythematosus（SLE）,such as renal disease or central nervous system symptoms.However,in some patients SCLE develops into SLE,causing progressive systemic damage over a period of years.Although the genetic and epigenetic factors that trigger SLE or the transition from SCLE to SLE remain unknown,studies indicate the importance of altered DNA methylation,especially hypomethylation in CD4+ T cells,in the pathogehesis of SLE.DNA methylation is an epigenetic regulator of several biological processes,including embryonic development,gene transcription,X chromosome inactivation,genomic imprinting and chromatin modification.Methylation involves the transference of a methyl group from the methyl donor S-adenosyl-L-methionine to the 5th carbon position in the cytosine ring of deoxycytosine（dC） bases in CG pairs. Methylation of CpG islands within promoter or enhancer regions suppresses transcription of target genes.Patterns of DNA methylation are established and maintained by DNA methyl transferases（DNMTs）.In humans,the three enzymes DNMT1,DNMT3a,and DNMT3b are known to have DNA methyltransferase activity.DNMT3a and DMMT3b are responsible for de novo methylation and modify unmethylated DNA whereas DNMT1 is thought to be required for maintaining methylation patterns and acts on hemimethylated DNA.Methylated DNA is recognized by a conserved family of methyl-DNA binding domain（MBD） proteins,of which there are five known members in mammalian genomes:MBD1,MBD2,MBD3, MBD4,and MECP2.All MBDs share a common methyl CpG binding domain and associate with proteins that play active roles in regulating DNA methylation,heterochromatin formation and gene transcription.In CD4+T cells of patients with SLE,MBD2 and MBD4 mRNA expression are elevated,and it has been proposed that this plays an active role in compromising T cell methylation.Evidence linking aberrant DNA methylation with SLE has been accumulating over the past two decades.Drugs that inhibit methylation trigger lupus-like autoimmune responses in mice and human lymphocytes, and T cells from patients with active lupus have genome-wide decreases in deoxymethylcytosin and gene-specific hypomethylation.These methylation deficits correlate with upregulated expression of autoimmune-related genes such as CD11a,CD70,perforin and CD40 Ligand.Overexpression this genes inducing excessive B cell stimulation, autoreactive monocyte and macrophage killing and contribute to the disease pathogenesis.SCLE is characterized by clinical features distinct from those of SLE and the genetic and epigenetic factors causing the disorder are unknown. In this work,we set out to investigate whether T cell hypomethylation is a determinant of the pathogenesis of other,less severe forms of erythematosus lupus conditions such as SCLE.Firstly we investigated the global DNA methylation levels as well as the expression of DNMTs and MBDs in CD4+ and CD8+ T cells of patients with SCLE,secondly we compared the expression level of some autoimmune-related genes such as CD11a,CD70,perforin and CD40 Ligand in CD4+ and CD8+ T cells of patients with SCLE and normal controls,and lastly we investigated whether gene-specific hypomethylation induces autoimmune-related genes overexpression in SCLE CD4+ T Cells.Part one Abnormal DNA methylation in T cells from patients with subacute cutaneous lupus erythematosusObjective To investigate the global DNA methylation and the expression of genes that regulate methylation in T cells of patients with SCLE.Methods PBMC（peripheral blood mononuclear cells） cells were isolated from the peripheral venous blood of 12 SCLE patients and 9 healthy donors by density gradient centrifugation.CD4+ and CD8+T cells were isolated from the PBMC using magnetic cell separation technique.We quantified global methylcytosine levels in CD4+ and CD8+T cells from patients with SCLE and healthy controls using the Methylamp^TM Global DNA Methylation Quantification Kit.mRNA levels of DNA methyltransferases（DNMTs） and methylated CpG binding proteins（MBDs） were measured by real-time quantitative polymerase chain reaction（RT-PCR）.Results Global DNA methylation was significantly reduced in CD4+ T cells from SCLE patients relative to controls（P=0.002）.No statistically significant differences were found between patients and controls when the methylation status of CD8+ T cells was assessed（p=0.231）.DNMT1 and DNMT3a mRNA levels were significantly lower in CD4+T cells from SCLE patients than that in controls（P=0.027;P=0.004, respectively）.MBD1,MBD3 and MBD4 mRNA levels were significantly higher in CD4+ T cells from SCLE patients compared to controls （p＜0.001;p＜0.001 and p=0.001,respectively）.Additionally,MECP2 and MBD4 mRNA expression was also significantly increased in CD8+ T cells from SCLE patients than that in controls（p=0.001;p=0.001, respectively）.We also found DNMT1 mRNA expression and CD4+T cell DNA methylation to be positive correlated within our SCLE patient cohort（r=0.590,P=0.044）.Conclusion These data suggest that globe DNA hypomethylation in CD4+T cells contributes to the development of SCLE. Part two Abnormal expression and methylation pattern of regulatory sequences of some autoimmune-related genes in T cells from patients with subacute cutaneous lupus erythematosusObjective To investigate the expression levels and methylation patterns of some autoimmune-related genes in CD4+ T cells from SCLE patients.Methods PBMC（peripheral blood mononuclear cells） cells were isolated from the peripheral venous blood of 12 SCLE patients and 9 healthy donors by density gradient centrifugation.CD4+ and CD8+T cells were isolated from the PBMC using magnetic cell separation technique.Using the flowcytometry to detect the expression levels of CD11a,CD70,CD40L and Perforin in CD4+ and CD8+T cells,mRNA levels of CD11a,CD70,CD40L and Perforin were measured by real-time quantitative polymerase chain reaction（RT-PCR）.Perforin protein was analysed by western-blot.Bisulfite sequencing was used to determine the methylation status of the regulatory sequence in which gene the expression are significantly difference.Results CD70 and Perforin is overexpressed on SCLE CD4+T cells. Demethylation of the CD70 and Perforin promoter regulatory sequence were seen in CD4+T cells from patients with SCLE.Conclusion Demethylation of regulatory sequence contributes to CD70 and Perforin overexpression in SCLE CD4+T cells.更多还原