摘要
目的：探讨1型糖尿病患者CD4+T细胞组蛋白修饰情况(组蛋白3赖氨酸4(H3K4)甲基化,组蛋白3赖氨酸9(H3K9)甲基化,组蛋白3(H3)乙酰化,组蛋白4(H4)乙酰化水平),并研究其组蛋白修饰酶mRNA表达水平。
方法：收集1型糖尿病患者16例,男9例,女7例,平均年龄33.1(15.0-63.0)岁；收集健康对照16例,男9例,女7例,平均’年龄33.6(20.0-60.0)岁。采用密度梯度离心法(Ficoll)分离外周血单个核细胞,用免疫磁珠进一步分离外周血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。实时荧光定量PCR法检测CD4+T细胞组蛋白甲基转移酶SET1, SUV39H1和SUV39H2,组蛋白乙酰转移酶P300和CREBBP,组蛋白去甲基化酶LSD1,组蛋白去乙酰化酶HDAC1, HDAC2, HDAC5, HDAC7和SIRT1基因mRNA表达水平。
结果：与健康对照组比较,1型糖尿病患者组CD4+T细胞组蛋白H3K9甲基化水平降低(P<0.05),H3K4甲基化水平有降低趋势,但无统计学意义(P>0.05),组蛋白H3乙酰化水平降低(P<0.01),而H4乙酰化水平无明显改变。1型糖尿病患者组外周血CD4+T细胞组蛋白H3K4甲基化,H3K9甲基化及H3乙酰化水平与空腹血糖,餐后血糖,糖化血红蛋白,空腹C肽和餐后C肽均无明显相关性。实时定量PCR结果显示1型糖尿病患者组CD4+T细胞SET1, SUV39H1, SUV39H2, P300, CREBBP, LSD1, HDAC1, HDAC2, HDAC5, HDAC7和SIRT1基因表达分别是健康对照组的-4.8,-7.6,-7.2,-1.9,-4.4,-1.6,-2.5,-2.0,-1.1,-3.4,-1。1倍。
结论：1型糖尿病患者外周血CD4+T细胞组蛋白修饰呈现异常,表现为H3K9甲基化水平和H3乙酰化水平降低；1型糖尿病患者外周血CD4+T细胞SET1,SUV39H1, SUV39H2, CREBBP, HDAC1, HDAC2和HDAC7基因表达显著下调。
Objective:To study histone modification patterns in CD4+T lymphocytes from type 1 diabetic patients, including histone 3 lysine 4 (H3K4) methylation, histone 3 lysine 9 (H3K9) methylation, histone 3 (H3) acetylation and histone 4 (H4) acetylation, and investigate associated histone modification enzymes'gene expression. Intended to provide novel epigenetic insights into the pathogenesis of type 1 diabetes.
Methods:We enrolled 32 volunteers into two groups, the first with 16 patients having a diagnosis of type 1 diabetes (9 male,7 female, median 33.1 years, range 15.0-63.0 years)and the second with 16 healthy volunteers (9 male,7 female, median 33.6 years, range 20.0-60.0 years). Peripheral blood mononuclear cells (PBMC) were isolated by the Ficoll method. The CD4+T cells were positively selected by magnetic beads according to the manufacturer's protocol. Histone extraction and detection of global H3K4/H3K9 methylation and histone H3/H4 aetylation were performed following the protocol of Epigentek assay kit. Furthermore, the mRNA levels of selected histone methyltransferases (SET1, SUV39H1 and SUV39H2), histone acetyltransferases (P300 and CREBBP), histone demethylase (LSD1) and histone deacetylases (HDAC1, HDAC2, HDAC5, HDAC7, SIRT1) were determined by using one step real-time quantitative PCR (TaqMan).
Results:Reduced global H3K9 methylation(P<0.05) and H3 acetylation(P<0.01) was observed in CD4+T cells of type 1 diabetes patients relative to healthy controls. There was a trend showing reduced H3K4 methylation, but with no statistical significance. However, H4 acetylation lever showed no difference between patients and controls. Type 1 diabetes CD4+T cells H3K4 methylation, H3K9 methylation and H3 acetylation were not related with fasting blood glucose (FBS), postprandial blood glucose (PBS), glycosylated hemoglobin (HbA1c), fasting C-peptide (FCP) and postprandial C-peptide (PCP). A part of selected histone modification enzymes mRNA expression were significantly downregulated in CD4+T cells of type 1 diabetes patients relative to healthy controls, the difference in SET1, SUV39H1, SUV39H2, P300, CREBBP, LSD1, HDAC1, HDAC2, HDAC5, HDAC7 and SIRT1 gene expression in patients compared with healthy controls was-4.8-fold,-7.6-fold,-7.2-fold,-1.9-fold,-4.4-fold,-1.6-fold,-2.5-fold,-2.0-fold,-1.1 fold,-3.4fold,-1.1-fold.
Conclusion:Abnormal histone modification patterns including reduced H3K9 methylation and H3 acetylation were observed in CD4+T cells from type 1 diabetes; SET1, SUV39H1, SUV39H2, CREBBP, HDAC1, HDAC2 and HDAC7 gene expression were significantly downregulated in CD4+T cells from type 1 diabetes patients.
引文
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