【作者】 彭亮；

【导师】 周宏灏；

【作者基本信息】 中南大学 ， 遗传药理学， 2007， 硕士

【摘要】 目的：本研究旨在从信号转导和代谢酶两方面揭示苦参碱的抗肿瘤机制，并初步讨论两者之间的相关性。STAT3经证实是与肿瘤发生发展相关的重要信号转导因子，其在90％的肿瘤细胞中都表现为异常增高。它的激活能够导致下游的癌基因如c-myc，BCL-_XL等表达增加，从而导致细胞癌变。本文对苦参碱能否抑制STAT3表达及功能作了研究。而STAT3经过复杂的网络调控系统和药物代谢酶的表达存在关联，本文研究了苦参碱对与肿瘤相关的药物代谢酶CYP1A1，CYP2D6，CYP2E1，CYP3A4，UGT1A1，UGT2B，UGT2B4的表达的影响。并对苦参碱对表达最多的代谢酶CYP3A4的活性影响进行探讨。并且初步探讨这些酶的表达与STAT3的关系。方法：培养HepG2细胞，使用0，0.125，0.25，0.5，1，2mM苦参碱，平行使用0，6.25，12.5，25，50，100ng／ml IL-6，观察药物作用后24h和48h后HepG2细胞中STAT3的mRNA和蛋白表达，药物作用后12h和24h P-STAT3的蛋白表达。观察HepG2细胞中CYP1A1，CYP2D6，CYP2E1，CYP3A4，UGT1A1，UGT2B，UGT2B4的表达，CYP3A4的蛋白表达及活性。mRNA表达使用半定量PCR检测，测蛋白表达使用western-blot。使用咪哒唑仑做探药，MS-HPLC检测咪哒唑仑及其代谢产物1-OH咪哒唑仑，考察CYP3A4的活性。结果：苦参碱作用后48h能够显著抑制STAT3的表达，2mM苦参碱使mRNA降低30％（P=0.001），蛋白表达降低50％（P=0.016）。而苦参碱对STAT3的激活也有抑制效果，P-STAT3的表达在12h就抑制了70％（P=0.001），不过在24h回落至50％左右（P=0.042）。苦参碱对CYP2E1，UGT1A1，UGT2B的表达都有诱导作用。2mM作用后48h，CYP2E1的表达为空白的1.5倍。作用后24h，UGT1A1，UGT2B表达分别为空白的2和2.5倍（P＜0.01）。苦参碱对CYP3A4的mRNA抑制在48h达到60％，蛋白表达被抑制80％，而活性更是达到90％以上（P＜0.01）。而IL-6对CYP2E1，UGT1A1，UGT2B的作用与苦参碱完全相反，可以抑制它们的表达达到70％左右。IL-6对CYP3A4也是抑制作用，抑制率达到80％。相关性分析表明，CYP2E1，UGT1A1，UGT2B与STAT3的表达相关系数（R²）分别为0.77，0.38，0.43，具有统计学意义。而与CYP3A4的表达和活性相关性不大（P＞0.05）。结论：苦参碱是一种能够抑制STAT3表达及活性的抗肿瘤药物。它能够抑制CYP3A4，诱导UGT1A1，UGT2B的表达，说明苦参碱能够抑制某些前致癌物的激活，增强细胞的解毒能力，起到保护细胞的作用。STAT3的激活剂IL-6对CYP2E1，UGT1A1，UGT2B的作用与苦参碱恰恰相反，相关性分析也表明这些酶的表达与STAT3的表达和激活显著相关。但是对CYP3A4的调节可能与STAT3关系不大，或者STAT3不是主要调节因素。更多还原

【Abstract】 AIMS: To investigate the anti-tumor mechanisms of matrine which including signal transduction and the expression of some drug metabolism enzymes (DMEs), and the relationship of STAT3 and DMEs.METHODS: Cultured the HepG2 cells with 0, 0.125, 0.25, 0.5, 1, 2mM Matrine or 0, 6.25, 12.5, 25, 50, 100ng/ml IL-6 for 12, 24 or 48h. then to detect the mRNA expression of STAT3 and both the protein expression of STAT3 and P-STAT3. we evaluated the CYP3A4 activity used MS-HPLC, assayed the mRNA expression of CYP450s, UGTs and stat3 by RT-PCR, detected the protein expression of CYP3A4, stat3, p-stat3 with Western-Blot.RESULTS: Matrine can significantly inhibit the expression and activation of STAT3. Matrine increased the mRNA expression of CYP2E1, UGT1A1, UGT2B and ecreased CYP3A4. 2mM Matrine increase the mRNA expression of CYP2E1, UGT1A1, UGT2B by about 10%, 90% and 20%, respectively. It also decreased CYP3A4 mRNA by about 60%, the protein expression of CYP3A4 by 80% and the activity of CYP3A4 by 90%, after treated with 2mM Matrine for 48h. 100ng/ml IL-6 can reduce the mRNA expression of CYP2E1, CYP3A4, UGT1A1, UGT2B for about 70%, and we found out the protein expression of CYP3A4 decreased 80% and the activity of CYP3A4 decreased 90%. The dependablity analysis indicated that the expression of CYP2E1, UGT1A1, UGT2B have significant association with STAT3 (R~2=0.77, 0.38, 0.43 respectively). All of these data has statistical significance.CONCLUSIONS: Matrine has an intense inhibition effect on the expression and activity of STAT3, which may be an important way for matrine’s anti-tumor effects. It also increased the mRNA expression of CYP2E1, UGT1A1, UGT2B and decreased CYP3A4, inhibited the protein expression and activity of CYP3A4. and IL-6 have an opposite influence on CYP2E1, UGT1A1, UGT2B except of CYP3A4. It indicated that matrine may induce CYP2E1, UGT1A1, UGT2B through Stat3.更多还原