【作者】 李亚萍；

【导师】 陆前进；

【作者基本信息】 中南大学 ， 皮肤性病学， 2009， 博士

【摘要】 系统性红斑狼疮(systemic lupus erythematosus,SLE)是一种多器官、多系统受累的自身免疫性疾病。其确切的病因及发病机理至今尚不十分清楚。近年来的研究结果表明T细胞DNA甲基化异常在SLE发病中起十分重要的作用。DNA甲基化是表观遗传学的重要调节机制。基因调节序列甲基化抑制基因转录,而低甲基化则激活基因转录。已有研究表明在体外抑制T细胞DNA甲基化,使T细胞甲基化敏感基因调控序列特定区域低甲基化,从而导致基因过度表达。这些基因包括CD11a(ITGAL)、perforin(PRF1)、CD70(TNFSF7)及CD40 ligand(TNFSF5)。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,在动物体内能诱导狼疮样疾病。而且上述甲基化敏感基因在SLE患者CD4~+T细胞过度表达,基因调控序列特定区域甲基化水平低下,处于低甲基化状态。因此,SLECD4~+T细胞的自身反应性与基因调控序列低甲基化密切相关。目前虽然DNA甲基化转甲基酶机制研究得比较清楚,但是DNA去甲基化的功能和机制仍然属于生物学倍受争议的研究领域。最新的研究结果显示Gadd45a(Growth arrest and DNA-damage-inducible protein 45 alpha,即生长停滞与DNA损伤诱导蛋白)可通过促进DNA修复,去除甲基化标记,从而减少表观遗传基因的沉默。紫外线照射可以诱导Gadd45a表达上调,同时紫外线照射可诱发和加重SLE发病,SLE患者T细胞基因组DNA甲基化水平降低。因此,我们提出SLE发病机理之一可能为:核蛋白Gadd45a过度表达→阻抑T细胞基因调控序列甲基化→T细胞甲基化敏感基因过度表达→杀伤自身巨噬细胞或辅助B细胞产生大量自身抗体→SLE。这一假定的发病机制将通过三个部分的研究来证实:1.Gadd45a在SLE CD4~+T细胞中的表达及与自身免疫的关系;2.紫外线对Gadd45a表达和甲基化水平的影响;3.诱导或抑制Gadd45a的表达对正常人CD4~+T细胞或SLE CD4~+T细胞甲基化敏感基因的表达、调控序列甲基化状态以及自身免疫反应的影响;通过这些研究将进一步揭示导致SLE T细胞基因病理性低甲基化及自身免疫反应的关键环节和分子机制,为SLE治疗寻找有效的新途径提供理论依据。第一部分SLE患者CD4~+T细胞Gadd45a的表达目的:探讨Gadd45a在SLE CD4~+T细胞中的表达及与自身免疫反应的关系。方法:采用Real-time RT-PCR、Western blotting的方法分别从mRNA及蛋白水平检测17例SLE患者和10例正常人CD4~+T细胞中Gadd45a基因的表达,同时检测细胞基因组DNA总体甲基化水平,并分析二者之间的相关性及其与疾病活动性评分(SLEDAI)的相关性。结果:和正常人比较,SLE患者CD4~+T细胞Gadd45a mRNA和蛋白水平均显著升高(P＜0.01或P＜0.05),细胞基因组DNA总体甲基化水平显著降低(P＜0.05),SLE患者CD4~+T细胞Gadd45a mRNA水平和SLEDAI评分呈显著正相关(R=0.568,P＜0.05);与细胞基因组DNA总体甲基化水平呈显著负相关(R=-0.676,P＜0.01)。结论:SLE患者CD4~+T细胞Gadd45a表达显著升高,且与DNA低甲基化及疾病活动性关联。第二部分紫外线对Gadd45a表达和DNA甲基化水平的影响第一节紫外线对Jurkat细胞Gadd45a表达和DNA甲基化水平的影响目的:研究紫外线对Jurkat细胞Gadd45a表达和DNA甲基化水平的影响。方法:分别以紫外线UVB 1.0J/cm~2和1.5J/cm~2照射Jurkat细胞后,于6小时、12小时、24小时和48小时收集细胞,采用Real-time RT-PCR法检测Gadd45a mRNA水平和甲基化敏感基因CD11a,CD70 mRNA水平,同时检测Jurkat细胞基因组DNA总体甲基化水平的变化。结果:(1)紫外线UVB1.0J/cm~2照射后6小时,Jurkat细胞Gadd45amRNA水平显著升高(P＜0.01),总体甲基化水平显著降低(P＜0.05),CD11a,CD70 mRNA水平也升高,但差异无统计学意义(P＞0.05)。照射后12小时Jurkat细胞Gadd45a CD11a,CD70 mRNA水平均显著升高(P＜0.05),Jurkat细胞总体甲基化水平显著降低(P＜0.05)。照射后24小时和48小时,Jurkat细胞Gadd45a mRNA水平升高,CD11a,CD70 mRNA水平升高,基因组DNA总体甲基化水平降低,但差异无统计学意义(P＞0.05)。(2)紫外线UVB1.5J/cm2照射后6小时、12小时、24小时和48小时,Jurkat细胞Gadd45a,CD11a,CD70 mRNA水平均显著升高(P＜0.05或P＜0.01),基因组总体甲基化水平显著降低(P＜0.05或P＜0.01)。结论:紫外线能诱导Jurkat细胞Gadd45a表达上调,同时甲基化敏感基因CD11a,CD70 mRNA表达升高,Jurkat细胞基因组DNA低甲基化。第二节紫外线对CD4~+T细胞Gadd45a表达和DNA甲基化水平的影响目的:研究紫外线对正常人CD4~+T细胞Gadd45a表达和DNA甲基化水平的影响。方法:免疫磁珠法从3例正常人外周血中分离CD4~+T细胞,紫外线UVB 1.5J/cm~2照射正常人CD4~+T细胞后,分别于6小时、24小时、48小时收集细胞,采用Real-time RT-PCR法检测Gadd45a mRNA,甲基化敏感基因CD11a,CD70 mRNA的表达,采用Western blotting法检测Gadd45a蛋白水平,同时检测CD4~+T细胞基因组DNA总体甲基化水平的变化。结果:紫外线照射后6小时、24小时、48小时的CD4~+T细胞Gadd45a,CD11a,CD70 mRNA水平均显著升高(P＜0.05),且Gadd45a蛋白表达显著升高(P＜0.05),基因组DNA总体甲基化水平降低,第48小时总体甲基化水平显著降低,差异有统计学意义(P＜0.05)。结论:紫外线能诱导CD4~+T细胞Gadd45a表达上调,同时甲基化敏感基因CD11a,CD70 mRNA表达升高,细胞基因组DNA总体甲基化水平下调。第三部分Gadd45a表达异常与自身免疫关系的研究第一节Gadd45a过表达诱导自身免疫反应目的:诱导Gadd45a过表达对正常人CD4~+T细胞甲基化敏感基因的表达、调控序列甲基化状态以及自身免疫反应的影响。方法:构建Gadd45a表达质粒PCDNA3.1-Gadd45a,电穿孔法瞬时转染正常人CD4~+T细胞,采用Real-time RT-PCR法检测甲基化敏感基因CD11a,CD70 mRNA的表达,ELISA法检测细胞增殖活性、自身B细胞IgG抗体产量,试剂盒检测细胞基因组DNA总体甲基化水平及亚硫酸氢钠基因组测序法检测CD11a(ITGAL)序列特异性甲基化状态的改变。结果:转染PCDNA3.1-Gadd45a的CD4~+T细胞Gadd45a,CD11a,CD70 mRNA水平升高,细胞增殖活性及自身B细胞的IgG抗体产量增加,总体甲基化水平降低,差异均有显著统计学意义(P＜0.05)。重亚硫酸氢钠测序结果显示ITGAL启动子调控序列平均甲基化水平显著降低(P＜0.01),七个CG位点中有六个位点(-1263,-1255,-1191,-1159,-1121,-1111bp)发生了低甲基化。结论:Gadd45a过表达可诱导正常CD4~+T细胞发生低甲基化,与自身免疫密切相关的甲基化敏感基因表达升高,细胞产生自身免疫性。第二节下调Gadd45a抑制自身免疫反应目的:抑制Gadd45a表达对SLE CD4~+T细胞甲基化敏感基因的表达、调控序列甲基化状态以及自身免疫反应的影响。方法:设计针对Gadd45a的小干扰RNA,电穿孔法瞬时转染活动性SLE患者的CD4~+T细胞,采用Real-time RT-PCR法检测Gadd45amRNA,甲基化敏感基因CD11a,CD70 mRNA的表达,流式细胞仪检测CD11a,CD70蛋白水平,ELISA法检测细胞增殖活性和IgG抗体产量。同时检测细胞基因组DNA总体甲基化水平及ITGAL特异性序列甲基化状态的改变。结果:转染后CD4~+T细胞Gadd45a,CD11a,CD70 mRNA水平降低,CD11a染色细胞百分率减少,CD70平均荧光强度降低,细胞增殖活性及自身B细胞的IgG抗体产量下降(P＜0.05)。细胞基因组DNA总体甲基化水平升高,但差异无显著性。重亚硫酸氢钠测序结果显示CD11a(ITGAL)启动子特异性序列平均甲基化水平显著升高(P＜0.05),七个CG位点中有四个位点(-1263,-1223,-1159,-1121)发生了高甲基化。结论:抑制Gadd45a的表达可逆转SLE患者CD4~+T细胞的异常低甲基化,下调与自身免疫密切相关的甲基化敏感基因表达,抑制自身免疫反应。更多还原

【Abstract】 Systemic lupus erythematosus(SLE) is a autoimmune disease which involved multiple organ systems.Its etiology and pathogenesis is unclear. Recent studies have shown that the T cell DNA demethylation play an important role in the pathogenesis of SLE.DNA methylation is one of many epigenetic factors that are important for cellular differentiation,gene regulation and genomic imprinting.In general,DNA hypomethylation is associated with gene transcription activation.Studies from Lu et al.showed that inhibiting T cell DNA methylation in vitro causes demethylation of regulatory sequences,contributes to increasing gene promoter activity,and thus leads to increasing gene expression.These genes include CD11a(ITGAL),perforin(PRF1),CD70(TNFSF7) and CD40 ligand(TNFSF5).Overexpression of these genes results in T cells autoreactivity,spontaneous monocyte/macrophage killing,B cell immunoglobulin overproduction.DNA methylation inhibitors could induce lupus-like autoimmunity in vitro and in vivo.Furthermore,T cells from patients with active lupus have genome-wide decrease in deoxymethylcytosin,and gene-specific hypomethylation.DNA hypomethylation induces autoimmune-related genes overexpression,and causes autoreactive monocyte/macrophage killing and excessive B cell stimulation.DNA demethylation also contributes to the striking female predilection of this disease.The mechanisms of demethylation in lupus T cells remain unknown.Recent study showed that Gadd45a can promote DNA repair,remove methylation marks,thereby reduce epigenetic silence of genes.Ultraviolet irradiation can upregulate expression of Gadd45a, and make SLE condition worse.Therefore,we propose the pathogenesis of SLE as follows:overexpression of nuclear protein Gadd45a can inhibit methylation at regulatory sequence of T cell methylation-sensitive genes, result in upregulated expression of the genes,then leads to spontaneous monocyte/macrophage killing and overstimulation of IgG synthesis, finally causes SLE.To prove the hypothesis,we used the following approach:firstly,to investigate the expression of Gadd45a in SLE CD4~+ T cells;secondly,to study the effect of UV on Gadd45a expression and DNA methylation;and thirdly,to examine the effect of upregulated and downregulated expression of Gadd45a on normal or SLE CD4~+ T cells. These studies would further reveal the mechanisms of demethylation of T cell genes in lupus,and provide a theoretical basis for more effective therapy of SLE. PartⅠExpression of Gadd45a in CD4~+ T cells from SLE patientsObjective:To investigate the expression of Gadd45a in SLE CD4~+T cells.Methods:Peripheral blood mononuclear cells(PBMCs) from 17 SLE patients and 10 healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation,CD4+ subsets were isolated by positive selection using Miltenyi beads.Expression of Gadd45a mRNA in CD4~+T cells was detected by real-time RT-PCR,and western blotting was used for protein expression in the same samples.The global methylation level was measured by DNA global methylation kit.The correlation between Gadd45a mRNA,global methylation level and systemic lupus erythematosus disease activity index(SLEDAI) were analyzed.Results:The expression of Gadd45a mRNA and protein in CD4+T cells from SLE patients were significantly elevated,Compared with controls(P＜0.01,P＜0.05,respectively).Global hypomethylation was observed in lupus CD4~+ T cells,compared with controls(P＜0.05).There was a significant negative correlation between Gadd45a mRNA expression and global methylation level(R=-0.676,P＜0.01).Furthermore, the Gadd45a mRNA level was positively correlated with increased disease activity in lupus patients as measured by SLEDAI(R=0.813,P ＜0.05).Conclusion:Gadd45a expression were significantly increased in lupus CD4~+ T cells,which is associated with demethylation and increased disease activity in SLE.PartⅡEffect of ultraviolet(UV) radiation on the expression of Gadd45a gene and methylation levels in Jurkat cells and CD4~+T cellsSectionⅠEffect of UV on the expression of Gadd45a gene and methylation levels in Jurkat cellsObjective:To explore the effect of UV on the expression of Gadd45a gene and methylation levels in Jurkat cells.Methods:Jurkat cells were irradiated with 1.0J/cm2 and 1.5J/cm2 UVB respectively,and collected at 6 hours,12 hours,24 hours and 48 hours after UVB irradiation.Real-time RT-PCR was used to detect the expression of Gadd45a mRNA and methylation sensitive genes CD11a, CD70 mRNA.Global methylation level was measured in the same samples. Results:(1) The expression of Gadd45a mRNA in Jurkat cells was significantly increased at 6 hours after UVB(1.0J/cm~2) irradiation (P＜0.01),accompanied with global demethylation((P＜0.05).The expression of CD11a,CD70 mRNA was upregulated,but the differences had no statistically significant.The expression of Gadd45a,CD11a,CD70 mRNA was signifcantly increased in Jurkat cells after 12 hours with irradiation UVB 1.0J/cm2(P＜0.05),and the global methylation level was significantly reduced(P＜0.05).24 hours and 48 hours after irradiation, Increased mRNA expression of Gadd45a,CD11a,CD70 mRNA and decreased global demethylation in Jurkat cells were also found at 24 and 48 hours after UVB(1.0J/cm~2) irradiation,but the differences had no statistically significant.(2) The expression of Gadd45a mRNA in Jurkat cells was significantly increased at 6,12,24 and 48 hours after UVB1.5 J/cm~2 irradiation,accompanied with significantly upregulated expression of CD11a,CD70 mRNA(P＜0.05 or P＜0.01).The global methylation level was significantly reduced at each time point after UVB1.5 J/cm~2 irradiation(P＜0.05 or P＜0.01).Conclusion:UVB can upregulate the expression of Gadd45a, CD11a and CD70 mRNA,and decrease global methylation in Jurkat cells SectionⅡEffect of UV on the expression of Gadd45a gene and methylation levels in CD4~+T cellsObjective:To explore the effect of UV on the expression of Gadd45a gene and methylation levels in CD4~+T cells.Methods:PBMCs from 3 healthy controls were isolated by Ficoll-Hypaque density gradient centrifugation,CD4~+ subsets were isolated by positive selection using Miltenyi beads.CD4~+T cells were irradiated with 1.5J/cm2 UVB,and collected at 6 hours,24 hours,48 hours after UVB irradiation.Real-time RT-PCR was used to detect the expression of Gadd45a mRNA and methylation sensitive genes CD11a, CD70 mRNA,while Gadd45a protein expression and global methylation level were measured in the same samples.Results:The expression of Gadd45a mRNA was significantly increased in CD4~+T cells at 6 hours,24 hours,48 hours after irradiation UVB 1.5 J/cm2(P＜0.05),accompanied with significantly upregulated expression of CD11a,CD70 mRNA(P＜0.05).Expression of Gadd45a protein was also upregulated.Meanwhile,the global methylation level decreased at 48 hours after irradiation(P＜0.05).Conclusion:UVB can upregulate expression of Gadd45a,CD11a and CD70,and reduce global methylation in CD4~+T cells. PartⅢAbnormal Gadd45a gene expression and autoimmunitySectionⅠInducing T cells autoreactivity by overexpressing Gadd45aObjective:To investigate the effect of overexpression of Gadd45a by transfecting Gadd45a expressed plasmid on expression and methylation status of autoimmunity related genes and autoreactivity in normal CD4~+ T cells.Methods:Gadd45a expressed plasmid(PCDNA3.1-Gadd45a) and control plasmid(PCDNA3.1+) were constructed,then transfected into normal human CD4~+ T cells by transient electroporation.Real-time RT-PCR was used to detect the expression of Gadd45a,CD11a and CD70 mRNA.Cell proliferation assay and detection of IgG antibody production were performed following the protocol of kits.The global methylation level and methylation status of ITGAL promoter regulatory elements were detected by the methylation kit and sodium bisulfite sequencing, respectively.Results:Compared with CD4~+ T cells transfected control plasmid The expression of Gadd45a,CD11a and CD70 mRNA was upregulated in CD4~+ T cells transfected with PCDNA3.1-Gadd45a(P＜0.05).Increased cell proliferation activity and enhanced IgG production,and accompanied with global demethylation were also found in CD4~+T cells transfected with PCDNA3.1-Gadd45a(P＜0.05).The average methylation of CpG pairs in ITGAL promoter sequence(-1263 to-1111) was significantly reduced(P＜0.01),and six out of seven CpGs(-1263,-1255, -1191,-1159,-1121,-1111) were significantly demethylated in CD4~+ T cells transfected with PCDNA3.1-Gadd45a.Conclusion:Overexpression of Gadd45a can upregulate expression of methylation sensitive genes,and increase autoreactivity in CD4+T cells.SectionⅡInhibiting T cells autoreactivity by downregulating Gadd45a expressionObjective:To investigate the effect of downregulating Gadd45a expression by transfecting Gadd45a-siRNA on expression and methylation status of autoimmunity related genes,and autoreactivity in SLE CD4~+T cells.Methods:Gadd45a-siRNA was transfected into SLE CD4~+T cells by transient electroporation.Real-time RT-PCR was used to detect the expression of Gadd45a and methylation sensitive genes CD11a and CD70 mRNA.CDlla and CD70 protein levels were measured by flow cytometry.Cell proliferation assay and production of IgG antibody were performed following the protocol of kits.The global methylation level and methylation status of ITGAL promoter regulatory elements were detected by the methylation kit and sodium bisulfite sequencing, respectively.Results:The expression of Gadd45a,CD11a and CD70 mRNA were significantly downregulated in SLE CD4~+ T cells transfected with Gadd45a-siRNA(P＜0.05).Percent of CD11a staining cells and CD70 MFI(mean fluorescence intensity) were decreased too.Reduced cell proliferation activity and IgG production,and accompanied with global hypermethylation were also found in SLE CD4~+ T cells transfected with Gadd45a-siRNA.The average methylation of CpG pairs at ITGAL promoter sequence(-1263 to-1111) was significantly increased (P＜0.05),and four out of seven CpGs(-1263,-1223,-1159,-1121) were significantly hypermethylated in SLE CD4~+ T cells transfected with Gadd45a-siRNA.Conclusion:Downregulation of Gadd45a expression can inhibit expression of methylation sensitive genes,and reduce autoreactivity in SLE CD4~+T cells.更多还原