论文部分内容阅读
目的:观察高糖刺激和甲基化抑制剂5-氮杂-2-脱氧胞苷(5-aza-dCyd)对人肾小球系膜细胞(HMCs)结缔组织生长因子(CTGF)基因启动子的甲基化水平和基因表达的影响。方法:用不同终浓度的5-aza-dCyd(0、1、2、5、10μmol/L)刺激HMCs,于48h提取细胞基因组DNA、总RNA和蛋白质;用不同浓度的葡萄糖(5 mmol/L、30 mmol/L)刺激细胞,于0、24、48、72 h收集细胞;应用甲基化特异性PCR(MSP)检测细胞CTGF基因启动子甲基化状态,RT-PCR检测CTGFmRNA表达,Western blot检测CTGF蛋白表达。结果:不同浓度5-aza-dCyd处理HMCs 48 h后,0、1、2、5μmol/L5-aza-dCyd组CTGF基因启动子呈半甲基化状态,均有微量CTGFmRNA和蛋白的表达,差异无统计学意义(均P>0.05);10μmol/L5-aza-dCyd组甲基化条带阴性,呈完全去甲基化状态,CTGF mRNA和蛋白表达均增加,与0μmol/L组比较差异有统计学意义(P<0.01)。5 mmol/L葡萄糖组HMCs各时间点CTGF基因启动子均呈半甲基化状态,表达微量CTGFmRNA和蛋白质,差异无统计学意义(均P>0.05);30 mmol/L葡萄糖组CTGF启动子在0 h呈半甲基化状态,甲基化条带较强;24 h甲基化条带减弱,48 h甲基化条带阴性,呈完全去甲基化状态,24 h、48 h、72 h CTGF mRNA和蛋白质表达均增加,与5 mmol/L组相应时间点比较差异有统计学意义(均P<0.05)。结论:高糖和甲基化抑制剂5-aza-dCyd均可诱导人肾小球系膜细胞CTGF基因启动子的去甲基化并增加CTGF的表达,提示DNA甲基化可能参与了人肾小球系膜细胞CTGF基因表达的调控。
AIM: To investigate the effects of 5-aza-2-dCyd, a high glucose-stimulated and methylated inhibitor, on the expression of connective tissue growth factor (CTGF) promoter of human mesangial cells (HMCs) The level of methylation and the effect of gene expression. Methods: HMCs were stimulated with different concentrations of 5-aza-dCyd (0,1,2,5,10 μmol / L), and genomic DNA, total RNA and protein were extracted at 48 hours. Different concentrations of glucose (5 mmol / L , 30 mmol / L), and the cells were harvested at 0, 24, 48 and 72 h. Methyl-specific PCR (MSP) was used to detect the promoter methylation status of CTGF gene. CTGF mRNA expression was detected by RT-PCR and Western Western blot was used to detect the expression of CTGF protein. Results: After treated with 5-aza-dCyd for 48 h, CTGF promoter in 0, 1, 2, 5 μmol / L 5-aza-dCyd group showed semi-methylation status with micro CTGF mRNA and protein expression, (P> 0.05). The methylation bands of 10μmol / L 5-aza-dCyd group were completely demethylated and the mRNA and protein expressions of CTGF were increased. Compared with 0μmol / L group, there was significant difference Statistical significance (P <0.01). The promoter of CTGF gene in 5 mmol / L glucose-treated HMCs were all semi-methylated, and showed no significant difference in the expression of CTGF mRNA and protein (all P> 0.05). CTGF promoter in 30 mmol / L glucose The methylated bands were weaker at 0 h, the methylated bands were stronger at 24 h, the methylated bands at 24 h were weaker, the methylated bands were negative at 48 h, and were completely demethylated at 24 h, 48 h and 72 h h CTGF mRNA and protein expression were increased, compared with the 5 mmol / L group corresponding time point difference was statistically significant (P <0.05). CONCLUSION: Both high glucose and methylation inhibitor 5-aza-dCyd can induce demethylation of CTGF gene promoter and increase CTGF expression in human mesangial cells, suggesting that DNA methylation may be involved in human kidney Regulation of CTGF gene expression in mesangial cells.