摘要
目的以人肝肿瘤细胞HepG2为工具细胞,通过优化选择毛细管区带电泳的运行电压、温度、缓冲溶液pH值等条件,建立细胞DNA氧化损伤后8OH dG的毛细管区带电泳分离检测方法,并以此方法分别检测未经H2O2处理组与经15mmol LH2O2处理组HepG2细胞DNA中8OH dG含量的变化。方法提取DNA采用饱和盐析法,避免经酚氯仿抽提导致8OH dG的产生。DNA溶液中加入DNaseI、蛇毒磷酸二酯酶及碱性磷酸酶得到游离核苷,并经去蛋白,二乙醚抽提后用于HPCE分析。优化后的分析条件为紫外检测器波长为254nm;pH值为9.5,浓度为20mmol L硼酸钠水溶液;毛细管长度为47cm;运行电压为25kV;温度为25℃;进样方式为重力进样,进样时间为20秒。结果在上述实验条件下,细胞DNA处理得到的游离核苷电泳结果与5种核苷标准品的电泳结果符合。经或未经H2O2处理组HepG2细胞DNA中均检测出8OH dG峰,H2O2处理组细胞DNA中8OH dG含量增加。结论此方法操作简便易行,进样量小,需时间及费用少,灵敏度较高,对人体无损害,并为8OH dG在人群生物监测方面的应用奠定了实验基础。
Objective To establish an optimized method to detect 8-OH-dG after DNA oxidation damage by capillary zone electrophoresis, Methods HepG2 cell was used as target cell and conditions for the separation and detection were obtained by studying the influence of pH of the running buffer, temperature, running voltage on the separation, 0 and 15mmol/L H2O2 were added into two groups of HepG2 cells（5 × 10^7 ） respectively for 24h. DNA was extracted by saturated salting out method to avoid the formation of additional 8-OH-dG by the method of phenol/chloroform extraction. DNA samples were digested to free nucleotides by incubation overnight at 37℃ with a mixture of DNase I, snake venom phosphoatase and alkaline phosphate. Proteins were removed and the supernatant was neutralized and then extracted with diethyl ether. The residue was evaporated to dryness and reconstituted and then analyzed under the optimized conditions by capillary zone electrophoresis. Results The optimized conditions were ： uncoated silica capillary （47cm × 50μm i. d. ）, 20mmol/L borate buffer （pH 9.5）, 25℃, 25kV. The sample was injected by hydrostatic method for 20s. In either of H2O2-treated group and H2O2-untreated group, the peak of 8-OH-dG was detected. The 8-OH-dG content of H2O2- untreated DNA increased. Conclusion The method is convenient, rapid, sensitive and cheap and safe. It provides an experimental platform to the application of 8-OH-dG in the biological monitoring of population.
出处
《卫生研究》
CAS
CSCD
北大核心
2005年第5期539-542,共4页
Journal of Hygiene Research
基金
国家自然科学基金资助项目(No.39990570)
