中文摘要：

目的利用反相高效液相色谱法检测MCF7细胞系中基因组DNA的甲基化水平。方法采用Nano LC（75μm×15 cm,5μm）和Micro LC（1.0 mm×15 cm,5μm）的C18反相色谱柱,以甲醇-醋酸铵缓冲液（pH 5.0）为流动相,使用线性梯度程序,将MCF7细胞中基因组DNA水解产物进行分离,两种体系流速分别是300 nl/min和40μl/min,在273 nm波长处检测,利用外标法分别检测DNA中脱氧胞嘧啶（dC）和甲基化脱氧胞嘧啶（5mdC）含量。结果 MCF7基因组DNA的水解产物,经反相色谱分离得到的峰图有较好的准确性与重复性;DNA水解后的产物为弱保留化合物,且Micro LC分离效果优于Nano LC;乳腺癌细胞MCF7甲基化水平为19.3%,高于正常细胞中基因组DNA甲基化水平。结论成功的将MCF7细胞中基因组DNA水解产物经反相色谱分离;使用Micro LC分离DNA水解后的弱保留产物较佳;MCF7细胞系基因组DNA甲基化水平高于正常细胞。

英文摘要：

Objective To detect the genomic DNA methylation in human breast cancer cell line MCF7 with reversed-phase high performance liquid chromatography（HPLC）.Methods The products of genomic DNA hydrolysis in MCF7 cells were chromatographed on Nano LC（75 μm×15 cm,5 μm）and Micro LC（1.0 mm×15 cm,5 μm）with ultraviolet detection at 270 nm,and eluted by the mobile phase of MeOH-NH4Ac（pH 5.0）at the flow rates of 300 nl/min and 40 μl/min under the condition of linear gradient program.The amounts of 2′-deoxycytidine（dC）and 5-methyl-2′-deoxycytidine（5mdC）were determined by external standard method.Results The products of DNA hydrolysis were successfully separated by reversed-phase HPLC with good repeatability and accuracy.The products were weakly retained and separated better by Micro LC.The level of global DNA methylation in MCF7 cells was 19.3%,which was higher than that in normal cells.Conclusion The products of DNA hydrolysis in MCF7 cells could be successfully separated by reversed-phase HPLC.Micro LC is superior to Nano LC in separating weak retention compounds of hydrolysis products.The level of DNA methylation is higher in MCF7 cells than in normal cells.