CAJ | 학술논문

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pH 9.6的48 mmol/L碳酸氢钠溶液[含60 mmoL/L十二烷基硫酸钠(SDS)]为分离缓冲液,检测波长为256 nm,在20 kV电压下,0.7 psi压力进样时间5 s,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、80 mmol/L)、pH值(9.4、9.6、9.8)、分离电压(15、17、19、20、22 kV)、进样时间(5、10、15、20、30 s)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10 min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、40 μmol/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.
목적 건립일충간편쾌속검측세포내정체DNA갑기화수평적방법,이용우림상갑기화진단.방법 채용고효모세관전영기술,이pH 9.6적48 mmol/L탄산경납용액[함60 mmoL/L십이완기류산납(SDS)]위분리완충액,검측파장위256 nm,재20 kV전압하,0.7 psi압력진양시간5 s,대2'-탈양포감(dC)、5-갑기-2'-탈양포감(mdC)、2'-탈양선감(dA)、2'-탈양흉감(dT)、2'-탈양조감(dG)5충물질진행분리.재차기출상검측안갑접령(MTX)유도적폐암A549내약세포주내정체DNA갑기화수평.결과 통과불단우화분리완충액중SDS농도(40、60、80 mmol/L)、pH치(9.4、9.6、9.8)、분리전압(15、17、19、20、22 kV)、진양시간(5、10、15、20、30 s)화모세관온도(15、20、25、30℃),건립고효모세관전영검측세포내정체DNA갑기화수평적방법,가재10 min내실현dC、mdC、dA、dT、dG적완전분리,기일내변이계수(CV)<0.2%,일간CV<2%,최저검출한위2μmoL/L.검측폐암A549친본세포갑기화수평위(4.80±0.52)%;이불동농도(15、30、40 μmol/L)MTX유도내약A549세포주적갑기화수평분별위(4.20±0.44)%、(3.70±0.36)%、(3.10±0.35)%.결론 건립고효모세관전영검측정체DNA갑기화수평적방법구유고효、쾌속、간단、령민적특점;MTX내약세포주내정체DNA갑기화수평수착내약농도적증가명현강저.
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.