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背景与目的:5-Fluorouracil(5-FU)主要通过抑制胸苷酸合成酶发挥抗癌作用,而二氢嘧啶脱氢酶是5-FU体内代谢的一个起始酶和限速酶。在本研究中,我们对二氢嘧啶脱氢酶、胸苷酸合成酶、c-myc和p53基因在小鼠肝脏中表达的昼夜节律进行研究。方法:本实验共采用了24只雄性B6D2F1小鼠。所有实验动物在实验中进行了4周光照同步化(12h光照:12h黑暗)。体温和休息-活动节律通过腹腔埋置的微型感受器进行动态监测。动物分别在3,7,11,15,19,23HALO(灯亮后小时数)被处死并取出肝脏冻存。肝脏标本中抽提的总RNA用LightCycler-SYBRGreenITM扩增系统进行实时定量RT-PCR。结果:小鼠的体温和休息-活动均呈现明显和相似的昼夜节律,其峰值均位于黑暗期(相当于小鼠活动相),谷值则位于光照期(相当于小鼠休息相)。二氢嘧啶脱氢酶在mRNA水平呈现出明显的昼夜节律性,其峰值时间位于16HALO左右即位于小鼠活动相(P=0.0012)。胸苷酸合成酶在mRNA水平具有昼夜节律表达的趋势,其峰值时间出现在光照期(P=0.079)。C-myc和p53在mRNA水平的节律性表达在本实验中无统计学意义。结论:二氢嘧啶脱氢酶和胸苷酸合成酶在mRNA水平呈现出昼夜节律性,二者峰值时间相差约12h,这一结果提示可以利用二者在转录水平的时间差异用5-FU进行时间化疗。
BACKGROUND & AIM: 5-Fluorouracil (5-FU) exerts its anticancer effect primarily by inhibiting thymidylate synthase, an enzyme that initiates and regulates 5-FU metabolism in vivo. In this study, we studied circadian rhythms of dihydropyrimidine dehydrogenase, thymidylate synthase, c-myc, and p53 genes expressed in mouse livers. Methods: Twenty-four male B6D2F1 mice were used in this study. All experimental animals were light-synchronized for 4 weeks in the experiment (12 h light: 12 h dark). Body temperature and rest - activity rhythms are dynamically monitored via intraperitoneal microcatheter. Animals were sacrificed at 3, 7, 11, 15, 19, 23 HALO (hours after lamp was illuminated) and the liver was removed and stored frozen. Total RNA extracted from liver specimens was subjected to real-time quantitative RT-PCR using the LightCycler-SYBR Green I TM amplification system. Results: The body temperature and rest-activity of mice showed obvious and similar circadian rhythms. The peaks were in the dark phase (equivalent to the mouse activity phase) while the troughs were in the light phase (equivalent to the mouse resting phase). Dihydropyrimidine dehydrogenase showed obvious circadian rhythms at mRNA level, and its peak time was around 16HALO, which was located in the mouse active phase (P = 0.0012). Thymidylate synthase has a tendency of circadian rhythm expression at the mRNA level, and its peak time appears in the light period (P = 0.079). The rhythmic expression of C-myc and p53 at the mRNA level was not statistically significant in this experiment. CONCLUSION: Dihydropyrimidine dehydrogenase and thymidylate synthase show circadian rhythms at mRNA level, with a difference of about 12 h in peak time. This result suggests that 5-FU can be used for the time difference of transcription level Time chemotherapy.