论文部分内容阅读
目的探讨DNA修复缺陷和癌基因高表达人胚肾上皮细胞转化模型在化学致癌物筛查中的应用价值。方法利用siRNA技术构建DNA损伤修复基因缺陷人胚肾上皮细胞(human embryonic kidney epithelial cell,HEK),采用微核实验检测DNA修复基因缺陷和H-rasV12癌基因高表达对DNA损伤修复功能的影响,并测定已知致癌物[硫酸镍(NiSO4)、N-甲基-N’-硝基-N-亚硝基胍(MNNG)]和促癌剂佛波酯(TPA)诱导上述细胞的转化活性,比较两种类型的HEK细胞模型筛查化学致癌物的应用价值。结果应用慢病毒介导的siRNA干扰技术成功构建了HEK-shERCC1、HEK-shERCC2、HEK-shATM和HEK-shMLH1等DNA损伤修复基因缺陷细胞株。与对照细胞HEK-shGFP相比,各DNA修复缺陷HEK细胞的生长速度、细胞形态和琼脂糖克隆的克隆形成率均没有差异,但HEK-sh ERCC2和HEK-shATM细胞对MMC(mitomycin C,MMC)所致遗传损伤的修复能力显著降低,1.0μg/ml MMC处理后,HEK-shERCC2和HEK-shATM细胞的微核率比对照细胞HEK-shGFP分别增加了34‰和30‰(P<0.05)。2μmol/L MNNG、400μmol/L NiSO4和800ng/ml TPA诱导HEKR细胞转化的时间分别是8周、8周和11周,但在染毒后20周均未能诱导各DNA损伤修复缺陷HEK细胞发生转化。结论癌基因高表达转化模型用于化学物质致癌活性的筛查,其灵敏性高于DNA修复基因缺陷细胞转化模型。
Objective To investigate the value of DNA repair defects and high expression of oncogenes in human embryonic kidney epithelial cells in the screening of chemical carcinogens. Methods Using siRNA technology to construct human embryonic kidney epithelial cells (HEK) with DNA damage repair gene defects, micronucleus assay was used to detect the effect of DNA repair gene defects and H-rasV12 oncogene high expression on DNA damage repair function. And determination of the known carcinogens [NiSO4, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG)] and the carcinogen phorbol ester (TPA) induced the above-mentioned cell transformation activity , Compare the application of two types of HEK cell models for screening chemical carcinogens. Results The DNA damage repair gene-defective cell lines such as HEK-shERCC1, HEK-shERCC2, HEK-shATM and HEK-shMLH1 were successfully constructed using the lentivirus-mediated siRNA interference technique. Compared with the control cell HEK-shGFP, there was no difference in the growth rate, cell morphology, and clone formation rate of agarose clones in HEK-sh ERCC2 and HEK-shATM cells against MMC (mitomycin C, MMC). ) The repair capacity of the resulting genetic damage was significantly reduced. After treatment with 1.0 μg/ml MMC, the micronucleus rate of HEK-shERCC2 and HEK-shATM cells was increased by 34 ‰ and 30 比 compared with the control cells HEK-shGFP, respectively (P<0.05). . The time of HEKR cell transformation induced by 2μmol/L MNNG, 400μmol/L NiSO4 and 800ng/ml TPA was 8 weeks, 8 weeks and 11 weeks, respectively. However, HEKR cells could not be induced to repair the DNA damage-defective HEK cells after 20 weeks. Conversion. Conclusion The oncogene high expression transformation model was used to screen for carcinogenicity of chemical substances, and its sensitivity was higher than that of DNA repair gene-deficient cell transformation model.