中文摘要：

目的 建立细胞色素氧化酶（cytochrome P-450,CYP）3A4启动子报告基因并在孕烷X受体（pregnane X receptor,PXR）表达阳性的肿瘤细胞中检测其转录活性。方法 利用PCR扩增CYP 3A4的启动子区远端调控序列（-7836/-7208）和近端调控序列（-362/＋52）序列;将上述序列克隆至p GL3-Promoter载体上;利用萤光素酶报告基因系统检测CYP 3A4报告基因的活性。结果 在肝癌细胞系中,PXR的激动剂利福平能够剂量依赖性地诱导远端调控序列荧光素酶报告基因（R^2=0.92;P=0.003 4）和近端调控序列荧光素酶报告基因（R^2=0.95;P=0.011）的活性,EC50为（5.65±0.58）μmol/L和（8.91±1.12）μmol/L;PXR的拮抗剂酮康唑能够剂量依赖性地抑制利福平诱导的远端调控序列荧光素酶报告基因（R^2=0.92;P=0.003 4）和PXRELuc（R^2=0.92;P=0.003 4）活性,IC50为（0.55±0.08）μmol/L和（0.94±0.14）μmol/L。此外,多种化疗药物（PXR的潜在配体）能够在PXR阳性的肿瘤细胞系中诱导远端调控序列荧光素酶报告基因和近端调控序列荧光素酶报告基因的活性。结论 成功构建了CYP 3A4启动子报告基因,在此基础上建立了在不同肿瘤细胞中检测PXR转录活性的方法。

英文摘要：

Objective To construct the cytochrome P-450 （CYP） 3A4 luciferase reporters and test their transcription activity in the positive tumor cells expressed by pregnane X receptor （PXR）. Methods The sequences of XREM （-7836/-7208） and PXRE （-362/＋52） in CYP 3A4 promoter were amplified via PCR method, and then they were cloned into pGL3-Promoter vectors. The activity of CYP 3A4 reporters was measured by luciferase assay. Results The Rifampicin, which was the agonist of PXR, induced the activity of XREM-Luc （R^2=0.92; P=0.003 4） and PXRE-Luc （R^2=0.95; P=0.011） in a dose dependent manner, with the ECs0 value of 5.65 ± 0.58 p. mol/L and 8.91 ＋ 1.12 ~ mol/L, respectively. The Ketoconazole, which was the antagonist of PXR, inhibited the activity of XREM-Luc （R^2=0.92; P=0.003 4） and PXRE-Luc （R^2=0.92; P=0.003 4） induced by Rifampi~in in a dose dependent manner, with the ICso value of 0.55 ± 0.08 μ moFL and 0.94 ±0.14 pμ mol/L, respectively. In addition, the effect of some potential PXR＇s ligands on CYP 3A4 reporters could be examined in the cells expressed by PXR. Conclusion The construction of the CYP 3A4 reporters is successfully established, which lays foundation for further functional study of CYP 3A4.