毛细管电泳法检测耐米托蒽醌乳腺癌细胞系全基因组甲基化水平

目的： 建立对米托蒽醌(Mit)耐药的乳腺癌MCF-7细胞系，并探讨乳腺癌细胞产生耐药与其基因组甲基化水平之间的关系。方法：用0.005、0.010和0.020 μmol/L浓度的米托蒽醌染毒乳腺癌MCF-7细胞，建立对米托蒽醌不同耐药程度的MCF-7耐药细胞系，采用流式细胞术检测各染毒组MCF-7细胞内药物荧光强度D(755)值，确定Mit耐药细胞系的建立；提取各组细胞的DNA， 利用毛细管电泳法检测野生型对照组及各染毒组细胞全基因组DNA甲基化水平。结果：野生型对照组、0.005、0.010和0.020 μmol/L米托蒽醌染毒组的D(755)值依次为16.1±0.04、14.3±0.12、13.3±0.07和9.7±0.08，与野生型对照组相比，随着药物染毒浓度的增加，MCF-7细胞内药物的D(755)值逐渐减少，其中0.020 μmol/L染毒组较对照组显著减少，差异有统计学意义 (P<0.05)，表明细胞耐药性增强；而上述各组DNA甲基化水平依次为42.25%±0.64%、37.97%±1.18%、34.27%±0.14%、31.16%±0.80%，与野生型对照组相比，各染毒组细胞基因组DNA甲基化水平逐渐降低 (P<0.05)，且各染毒组间两两比较差异均具有统计学意义 (P均<0.05)。结论：成功建立了对Mit耐药的MCF-7细胞系；确定了毛细管电泳法检测基因组DNA甲基化水平的电泳分离条件。

mitoxantrone-resistant breastcancer cell lines by capillaryelectrophoresis

【ABSTRACT】 OBJECTIVE： To establish a mitoxantrone-resistant MCF-7 breast cancer cell line,and toexplore the relationship between breast cancer cells developing drug resistance and their genomic methylation levels.METHODS：Using cells infected with 0,0.005,0.010 and 0.020 μmol/L mitoxantrone,different levels of drugresistance of MCF-7/Mit cell line was established. Flow cytometer was used to detect the accumulation of mitoxantrone inMCF-7 cells to confirm drug resistant cell lines. Then,we used capillary electrophoresis to evaluate genomic DNAmethylation levels of wide type and cells with different degrees of resistance. RESULTS：Compared with the controlgroup,with increasing mitoxantrone concentration,drug accumulation gradually decreased in MCF-7 cells. In the wildtypecontrol group,0.005,0.010 and 0.020 μmol/L -treated groups the drug D（755） values were 16.1±0.04, 14.3±0.12,13.3±0.07 and 9.7±0.08,respectively. The 0.02 μmol/L exposure group was significantly reduced comparedwith the control group,indicating that drug resistance was significantly increased （P〈0.05）. In the wild-type controlgroup,0.005,0.010 and 0.020 μmol/L -treated groups,the DNA methylation levels were 42.25%± 0.64%,37.97%±1.18%,34.27%±0.14% and 31.16%±0.80%, respectively. Compared with the control group, genomic DNAmethylation levels in each group of treated cells gradually decreased （P〈0.05）,with significant differences between eachtwo groups （P〈0.05）. CONCLUSION：Mit drug-resistant MCF-7 cell line was successfully set up. The separationcondition of capillary electrophoresis when measuring genomic DNA methylation levels was determined.