氨甲蝶呤耐药细胞内整体DNA甲基化水平检测方法的建立及其应用  

Establishment and application of medical method for quantification of genomic DNA methylation in methotrexate drug resistance cells

作　　者：李明[1] 胡世莲 何晓东[1] 陶绍能[1] 董林[1] 朱园园[1] 吴剑锋[1] 沈佐君[1] 

机构地区：[1]安徽医科大学附属省立医院安徽省临床检验中心,合肥230001 [2]医学研究中心老年病研究室

出　　处：《中华检验医学杂志》2009年第2期204-208,共5页Chinese Journal of Laboratory Medicine

基　　金：基金项目：国家自然科学基金资助项目（30672011）;安徽省自然科学基金资助项目（050430902）;安徽省人才开发资金资助项目（20052040）

摘　　要：目的建立一种简便快速检测细胞内整体DNA甲基化水平的方法，以用于临床甲基化诊断。方法采用高效毛细管电泳技术，以pH9．6的48mmol／L碳酸氢钠溶液[含60mmoL／L十二烷基硫酸钠（SDS）]为分离缓冲液，检测波长为256nm，在20kV电压下，0．7psi压力进样时间5s，对2’-脱氧胞苷（dC）、5-甲基-2’-脱氧胞苷（mdC）、2’-脱氧腺苷（dA）、2’-脱氧胸苷（dT）、2’-脱氧鸟苷（dG）5种物质进行分离。在此基础上检测氨甲蝶呤（MTX）诱导的肺癌A549耐药细胞株内整体DNA甲基化水平。结果通过不断优化分离缓冲液中SDS浓度（40、60、80mmol／L）、pH值（9．4、9．6、9．8）、分离电压（15、17、19、20、22kV）、进样时间（5、10、15、20、30S）和毛细管温度（15、20、25、30℃），建立高效毛细管电泳检测细胞内整体DNA甲基化水平的方法，可在10min内实现dC、mdC、dA、dT、dG的完全分离，其日内变异系数（CV）〈0．2％，日间CV〈2％，最低检出限为2μmol／L。检测肺癌A549亲本细胞甲基化水平为（4．80±0．52）％；而不同浓度（15、30、40μmol／L）MTX诱导耐药A549细胞株的甲基化水平分别为（4．20±0．44）％、（3．70±0．36）％、（3．10±0．35）％。结论建立高效毛细管电泳检测整体DNA甲基化水平的方法具有高效、快速、简单、灵敏的特点；MTX耐药细胞株内整体DNA甲基化水平随着耐药浓度的增加明显降低。Objective To establish a rapid and convenient method for determination of genomic DNA methylation in cells. Methods Five standard substances （dC, mdC, dA, dT and dG） were separated by high-performance capillary electrophoresis. Bare fused-silica capillary was used and eletrophoresis buffer was 48 mmol/L NaHCO3 with 60 mmol/L SDS, pH 9. 6. The temperature of separation was controlled at 25℃ and a voltage of 20 kV was applied. The separation of the mixture was performed at a wavelength of 256 nm with UV-Vis detection and injection time was 5 seconds at 0. 7 psi. Under optimal condition, genomic DNA methylation in methotrexate drug-resistant A549 cells was detected. Results The optimal condition was made by adjusting SDS concentration（40, 60, 80 mmol/L）, pH value of running buffer（9.4, 9.6, 9.8）, voltage（15, 17, 19, 20, 22 kV）, injection time（5, 10, 15, 20, 30 s） and capillary temperature（15, 20, 25, 30 ℃ ）. The method for determination of genomic DNA methylation in cells was established. Five substances were completely separated by high-performance capillary electrophoresis in 10 mins. Intra-day coefficient of variation was less than 0. 2% and inter-day coefficient of variation was less than 2%. The minimal detection limit was 2 μmol/L. Percentage of mdC in A549 parent cells was （4. 80 ± 0. 52）%. Percentage of mdC in 15, 30, 40 μmol/L methotrexate drug-resistant A549 cells were （4. 20 ±0.44）%, （3.70 ±0.36）%, （3. 10 ±0.35）%, respectively. Conclusions Genomic DNA methylation can be quantificated by high-performance capillary electrophoresis efficiently, rapidly, conveniently and sensitively. Genomic DNA methylation in methotrexate drug resistance cells decreases significantly.

关 键 词：氨甲蝶呤 DNA甲基化 电泳 毛细管 

分 类 号：R686[医药卫生—骨科学]