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目的:探讨叶酸在N-甲基-N-硝基-N-亚硝基胍(MNNG)影响哈萨克族食管上皮细胞DNA甲基转移酶1(DNMT1)活性及表达中的作用,为食管癌的防治提供理论依据。方法:采用3因素3水平(3×3)析因设计将体外培养的哈萨克族食管上皮细胞分别暴露于不同浓度叶酸(0.000、0.400、0.800μg/m L)和MNNG(0.000、0.750、1.500μg/m L)的培养液中作用48、72和96 h后,采用ELISA法检测DNMT1酶活性;采用RT-PCR的方法检测DNMT1 m RNA表达水平;采用Western blot法检测DNMT1蛋白的表达水平。结果:不同浓度叶酸、MNNG作用细胞不同时间后,在固定MNNG浓度和时间因素下,随着叶酸浓度的增加DNMT1酶活性、DNMT1 m RNA和蛋白表达水平均逐渐降低(P均<0.01);固定叶酸浓度和时间,随着MNNG浓度的增加DNMT1酶活性、DNMT1 m RNA和蛋白表达水平均逐渐升高(P均<0.01);固定MNNG和叶酸浓度,随着干预时间的延长DNMT1酶活性、DNMT1 m RNA和蛋白表达水平均逐渐升高(P均<0.01);叶酸浓度与MNNG浓度之间、MNNG浓度与作用时间之间均存在交互作用(P<0.01)。不同叶酸浓度与不同MNNG浓度之间、不同叶酸浓度与不同作用时间之间、不同MNNG浓度与不同作用时间之间差异均有统计学意义(P<0.05)。结论:叶酸浓度降低对MNNG影响哈萨克族食管上皮细胞DNMT1酶活性及表达有一定的激发作用,对食管癌的发生有一定的促进作用,而保持充足的叶酸则对MNNG的损害起到一定的保护作用。
Objective: To investigate the effect of folic acid on the activity and expression of DNA methyltransferase 1 (DNMT1) in Kazakh esophageal epithelial cells induced by MNNG, Prevention provides a theoretical basis. Methods: Kazakh esophageal epithelial cells cultured in vitro were exposed to various concentrations of folic acid (0.000, 0.400, 0.800 μg / mL) and MNNG (0.000, 0.750, 1.500 μg / m L) for 48, 72 and 96 h. The DNMT1 activity was detected by ELISA. The expression of DNMT1 mRNA was detected by RT-PCR. The expression of DNMT1 protein was detected by Western blot. Results: Under different concentration of folic acid and MNNG, DNMT1 mRNA and protein expression gradually decreased with the concentration of folic acid and MNNG at a fixed time (P <0.01) Folic acid concentration and time, DNMT1, DNMT1 mRNA and protein expression gradually increased with the increase of MNNG concentration (all P <0.01). MNNG and folic acid concentrations were fixed. With the prolongation of intervention time, DNMT1 activity, DNMT1 (P <0.01). There was an interaction between folic acid concentration and MNNG concentration, MNNG concentration and action time (P <0.01). There was significant difference between different concentrations of folic acid and different concentration of MNNG, different concentrations of folic acid and different time of action, different concentrations of MNNG and different time of action (P <0.05). CONCLUSION: The decrease of folic acid has a certain stimulating effect on the activity and expression of DNMT1 in Kazakh esophageal epithelial cells induced by MNNG, which may promote the occurrence of esophageal cancer, and maintain sufficient folic acid to protect MNNG effect.