【作者】 侯粲；

【导师】 周智广；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 目的：探讨2型糖尿病(T2DM)患者外周血单个核细胞(PBMC)整体组蛋白修饰情况及PBMC肿瘤坏死因子-α(TNF-a)、环氧合酶-2(COX-2)基因启动子区域组蛋白乙酰化情况。方法：收集12例T2DM患者及12例健康对照PBMC,H3K4/H3K9甲基化及H3/H4乙酰化检测试剂盒提取组蛋白和检测H3K4/H3K9甲基化及H3/H4乙酰化水平。实时多聚酶链反应(Real time-PCR)法检测T2DM PBMC组蛋白修饰酶谱、TNF-a mRNA、COX-2 mRNA表达水平。染色质免疫沉淀技术检测TNF-a及COX-2基因启动子区域组蛋白H3乙酰化状态。结果：与健康对照组比较,T2DM患者PBMC组蛋白H3K4甲基化水平升高(P<0.05)、H3乙酰化水平升高(P<0.01)、H4乙酰化水平升高(P<0.05)。Real time-PCR结果显示T2DM患者PBMC组蛋白去乙酰化酶HDAC2、SIRT1 mRNA表达分别是健康对照组的3.55±0.30(P<0.01)、0.40±0.25倍(P<0.05)；组蛋白乙酰转移酶P300、CREBBP mRNA表达分别是健康对照组的3.11±0.38(P<0.05)、2.78±0.45(P<0.05)倍；组蛋白去甲基化酶KDM3A、KDM5B和LSD1 mRNA表达分别是健康对照组的0.43±0.43(P<0.05)、3.55±0.30(P<0.01)和0.36±0.35(P<0.05)倍；组蛋白甲基转移酶SET1mRNA表达是健康对照组的3.06±0.51倍(P<0.05)。与正常对照组比较,T2DM患者PBMC TNF-a及COX-2 mRNA表达增高,TNF-a及COX-2基因启动子区域组蛋白H3呈高乙酰化状态。结论：2型糖尿病PBMC整体组蛋白修饰及组蛋白修饰酶谱异常,TNF-a及COX-2基因启动子区域组蛋白H3高乙酰化。目的：探讨成人隐匿性自身免疫糖尿病(LADA)患者外周血CD4+T细胞整体组蛋白修饰及CD4+T细胞叉头框蛋白3 (Foxp3)基因启动子区域组蛋白乙酰化情况。方法：收集16例LADA患者及16例健康对照外周血单个核细胞,磁珠分选CD4+T细胞,H3K4/H3K9甲基化及H3/H4乙酰化检测试剂盒提取组蛋白和检测H3K4/H3K9甲基化及H3/H4乙酰化水平。Real time-PCR法检测CD4+T组蛋白修饰酶谱及Foxp3 mRNA表达水平。染色质免疫沉淀技术检测Foxp3基因启动子区域组蛋白H3乙酰化状态。结果：与健康对照组比较,LADA患者CD4+T细胞组蛋白H3/H4乙酰化水平降低(P<0.05)。Real Time-PCR结果显示LADA患者组CD4+T细胞组蛋白去乙酰化酶HDAC2、HDAC5、SIRT1 mRNA表达上调；组蛋白乙酰转移酶P300、CREBBP、PCAF mRNA表达下调；组蛋白去甲基化酶LSD1 mRNA表达是健康对照组的0.17±0.13(P<0.01)倍；组蛋白甲基转移酶SUV39H2 mRNA表达是健康对照组的0.36±0.11(P<0.05)倍。LADA患者外周血CD4+T细胞Foxp3 mRNA表达较正常对照组降低(P<0.05),Foxp3基因启动子区域(-501/-416)(-204/-114)组蛋白H3乙酰化水平降低(P<0.01)。结论：LADA患者CD4+T细胞整体组蛋白修饰及组蛋白修饰酶谱异常,Foxp3基因启动子区域(-501/-416)(-204/-114)组蛋白H3低乙酰化。目的探讨组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)干预对LADA患者CD4+T细胞乙酰化水平及Foxp3表达的影响。方法采用密度梯度离心法分离LADA患者外周血单个核细胞,免疫磁珠分离CD4+T细胞,分别给予TSA处理培养相应时间后收集细胞。Western-blot法分析TSA干预对LADA患者CD4+T细胞H3乙酰化及Foxp3表达影响。结果与正常对照组相比,TSA干预组LADA患者CD4+T细胞组蛋白H3乙酰化水平及Foxp3表达水平均增加,并呈时间和剂量依赖性。结论TSA干预增加LADA患者CD4+T细胞H3乙酰化水平及Foxp3表达水平。更多还原

【Abstract】 AimsTo investigate the global histone modification patterns and the status of histone H3 acetylation at Tumor necrosis factor-a (TNF-a) and cyclooxygenase-2 (COX-2) gene promoter region in peripheral blood mononuclear cells (PBMCs) collected from Type 2 diabetic patients.MethodsGlobal histone H3/H4 acetylation and H3K4/H3K9 methylation in PBMCs from 12 Type 2 diabetic patients and 12 healthy control subjects were quantified by the EpiQuikTM global histone H3/H4 acetylation and H3K4/H3K9 methylation assay kit. The differential expression of the histone modification enzymes and TNF-a mRNA, COX-2 mRNA were measured by real-time polymerase chain reaction (Real-time PCR). Chromatin immunoprecipitation analysis was used to detect the status of histone H3 acetylation at TNF-a and COX-2 gene promoter region.ResultsIncreased global H3K4 methylation (P<0.05), H3 acetylation (P <0.01) and H4 acetylation(P<0.05) was observed in PBMCs from type 2 diabetic patients compared with controls. mRNA level of the histone deacetylases HDAC2, SIRT1 in patients PBMCs was 3.55±0.30 (P <0.01),0.40±0.25 (P<0.05) fold that in healthy controls. mRNA level of the histone acetyltransferases P300, CREBBP in patients PBMCs was 3.11±0.38 (P<0.05),2.78±0.45 (P<0.05)fold that in healthy controls. mRNA level of the histone demethylase KDM3A, KDM5B, LSD1 in patients PBMCs was 0.43±0.43 (P<0.05),3.55±0.30 (P<0.01),0.36±0.35 (P<0.05) fold that in healthy controls. mRNA level of the histone methytransferases SET1 in patients PBMCs was 3.06±0.51 (P<0.05) fold that in healthy controls. TNF-a and COX-2 mRNA was overexpressed in PBMCs from Type 2 diabetic patients compared with normal controls. H3 acetylation at the TNF-a and COX-2 gene promoter region was elevated in PBMCs from Type 2 diabetic patients compared with normal controls.ConclusionAn aberrant expression pattern of histone modification and histone modification enzymes existed in PBMCs of T2DM. H3 acetylation at the TNF-a and COX-2 gene promoter region was elevated. AimsTo investigate global histone modification patterns and the status of histone H3 acetylation at Forkhead box protein 3 (Foxp3) gene promoter regions in CD4+ T cells collected from latent autoimmune diabetes in adults (LADA).MethodsGlobal histone H3/H4 acetylation and H3K4/H3K9 methylation in CD4+ T cells from 16 LADA patients and 16 healthy control subjects were analyzed by the EpiQuikTM global histone H3/H4 acetylation and H3K4/H3K9 methylation assay kits. The differential expression of the histone modification enzymes and Foxp3 mRNA were measured by Real-time PCR. Chromatin immunoprecipitation analysis was used to detect the changes of histone H3 acetylation at the promoter region of Foxp3 gene.ResultsReduced H3/H4 acetylation was observed in the LADA CD4+ T cells compared with the controls (P<0.05). Compared with healthy controls, mRNA levels of the histone deacetylases HDAC2, HDAC5 and SIRT1 in the CD4+T cells of patients were upregulated, mRNA levels of the histone acetyltransferases P300, CREBBP, PCAF in patients CD4+ T cells were downregulated. mRNA level of the histone demethylase LSD1 in patients CD4+ T cells was 0.17±0.13 (P<0.01) fold that in healthy controls. mRNA level of the histone methytransferases SUV39H2 in patients CD4+ T cells was 0.36±0.11 (P<0.05) fold that in healthy controls. Foxp3 mRNA was downregulated in CD4+ T cells from LADA patients (P<0.05). H3 acetylation at the Foxp3 gene promoter region (-501/-416) (-204/-114) was lower in CD4+ T cells from LADA patients compared with normal controls (P<0.01)ConclusionAn aberrant expression pattern of histone modification and histone modification enzymes existed in CD4+ T cells of LADA. H3 acetylation at the Foxp3 gene promoter region (-501/-416) (-204/-114) was decreased. Objective To investigate whether the histone deacetylase inhibitor, trichostatin A (TSA) interfere with H3 acetylation and Foxp3 expression in CD4+ T cells from LADA patients.Methods Peripheral blood mononuclear cells (PBMC) isolated from blood of healthy volunters were prepared using the Ficoll-Hypaque method. CD4+ T cells were isolated using magnetic beads. CD4+ T cells were treated with different concentrations of TSA. Cells were collected after appropriate time, H3 acetylation and the expression of Foxp3 was analyzed by Western-blot.Results Compared with control group, TSA intervention increased H3 acetylation and Foxp3 expression in CD4+ T cells from LADA patients time-dependantly and dose-dependantly.Conclusion H3 acetylation and Foxp3 expression in LADA CD4+ T cells was elaveted by TSA treatment.更多还原