中文摘要：

目的使用超高效液相色谱-串联质谱测定骨髓间充质干细胞中全基因组DNA甲基化水平,观察其衰老过程中甲基化率的变化。方法从骨髓间充质干细胞中提取DNA,使用酶解法将DNA分解成单个脱氧核苷,采用超高效液相色谱法分离,正离子电喷雾与多反应监测模式进行定量,分析5-甲基脱氧胞嘧啶核苷、脱氧鸟嘌呤核苷的相对含量,计算全基因组DNA甲基化率。结果对1μg DNA样本进行1 h酶解反应,2 min内可分析出5.00×10^-4水平以上的DNA甲基化率,日内及日间精密度分别在7.12×10^-2和0.119以内。随着骨髓间充质干细胞传代扩增培养,干细胞不断衰老,其甲基化率逐渐降低,传至P4-P6代时,干细胞甲基化率最低,随着传代的继续,其甲基化率逐渐上升。结论此方法成功应用于检测研究骨髓间充质干细胞在衰老过程中全基因组DNA甲基化水平的变化,得到了衰老影响骨髓间充质干细胞DNA甲基化的初步数据。经考察,该方法样本前处理方法简便,可操作性强,定量方式快速准确,具有良好的灵敏度与重复性。

英文摘要：

Aim To measure the methylation rate of bone marrow mesenchymal stem cells（ BMMSCs） using ultra-performance liquid chromatography-tandem mass spectrometry,and determine the methylation rate in the process of senescence. Methods The DNA extracted from BMMSCs would be digested into individual deoxynucleosides using enzymatic hydrolysis. To quantify the global genomic DNA methylation rate,we developed a method using ultra-performance liquid chromatography-tandem mass spectrometry with multiple reaction monitoring to simultaneously measure the levels of 5-methyl deoxycytidine and deoxyguanosine in digested genomic DNA. Results The DNA methylation rate could be analyzed within two minutes after hydrolyzing1μg DNA for an hour. The detection limit of DNA methylation rate was 5. 00 × 10^-4. The coefficient of variance of the intra-day and inter-day precision was within 7. 12 × 10^-2 and 0. 119,respectively. With the subculture of BMMSCs,the methylation rate gradually decreased. The methylation rate of stem cells was the lowest in 4 - 6 generation,and gradually increased with the subculture. Conclusions Using this method,the preliminary data on DNA methylation of BMMSCs in the process of senescence are obtained. The method is simple and convenient for sample preprocessing,and the method is fast and accurate with good sensitivity and repeatability.