过氧化氢诱导肠上皮干细胞DNA氧化损伤模型的建立

目的：探索建立过氧化氢诱导肠上皮细胞DNA氧化损伤模型，以便在体外进一步研究DNA氧化损伤在大肠癌发生发展中的作用。方法：过氧化氢以RPM11640培养基稀释成100、50、10、5、μmol／L5个浓度，以RPMI1640培养荆作空白对照。作用人胎肠上皮干细胞不同时间（10min、30min、1h、1．5h、12h、24h）后，以MTT法检测细胞生存状况，以免疫组织化学方法检测DNA特异性氧化损伤产物8-羟-2-脱氧鸟苷（8-OhdG），用SPSS10．0软件以线性回归方法检验肠上皮干细胞在不同时间的生存率变化趋势。结果：MTT活性检测结果表明，在10～30min时，各浓度过氧化氢作用后的细胞活性较高，为60％～80％，但随时间推移，细胞活性下降，至24h，降至30％左右，时间趋势检验表明，除100、50μmol／L过氧化氢组和空白对照组外，其余各组的生存率随过氧化氢作用时间的延长，生存率下降，时间趋势差异具统计学意义。过氧化氢作用时间在10～30min内，100、50μmol／L组的细胞活性明显低于1～10μmol／L组20％左右，但在1．0h以上各组，虽然过氧化氢浓度不同，但在同一作用时间内细胞活性无明显差别。过氧化氢处理肠上皮细胞15min后进行8-OhdG免疫组织化学检测，结果除对照组阴性外，其余各组肠上皮细胞在不同浓度过氧化氢作用后的胞核及胞浆染色均呈棕褐色，为阳性。结论：过氧化氢能诱导肠上皮细胞发生DNA氧化损伤，肠上皮细胞DNA氧化损伤模型的条件以1～10μmol／L过氧化氢作用10～30min为宜。

Establishment of DNA oxidative damage model in colorectal crypt cells by hydrogen peroxide

OBJECTIVE: To induce DNA oxidative damage in colorectal crypt cells by hydrogen peroxide in vitro. METHODS: Hydrogen peroxide was diluted into 100, 50, 10, 5 and 1 micromol/L with RPMI 1640. Colorectal crypt cells were treated with peroxide for 10 min, 30 min, 1 h, 1.5 h, 12 h and 24 h respectively. The survival of colorectal crypt cell was measured by MTT method, and the DNA oxidative damage special product, 8-OhdG was detected with immunohistochemical staining. Liner regression was used to measure the time trend of survival rate with SPSS 10.0 software. RESULT: Survival rate of colorectal crypt cell was 60% and 80% after 10 min of hydrogen peroxide treatment. The longer treatment of hydrogen peroxide, the lower survival rate; the survival rate was reduced to 30% in 24 h. After 10 or 30 min treatment of 100 or 50 micromol/L hydrogen peroxide, the survival rates of colorectal crypt cells were reduced by 20% compared with those of 10, 5 and 1 micromol/L hydrogen peroxide. However, while cells were treated with different concentrations of hydrogen peroxide for 1.0 h or above, there were no differences in cell survival rates. The time trend test results demonstrated that the survival rates of colorectal crypt cells treated with 10, 5 and 1 micromol/L hydrogen peroxide were significantly decreased with the time length of treatment. Colorectal crypt cells treated with different concentrations of hydrogen peroxide for 15 minutes were positively stained brown in cytoplasm and nuclear by immunohistochemistry with 8-OhdG monoclonal antibody. CONCLUSION: Hydrogen peroxide could induce DNA oxidative damage in colorectal crypt cells. And treated with 1 - 10 micromol/L hydrogen peroxide for 10 - 30 min, DNA oxidative damage is apt to be induced in colorectal crypt cell.