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目的建立基于人孕烷X受体(human pregnane X receptor,hPXR)的UGT1A1的报告基因模型,研究中药提取物通过人孕烷X受体途径对UGT1A1的潜在诱导能力。方法以人基因组DNA为模板扩增UGT1A1的远端和近端启动子,连接到pGL3载体上,构建pGL3-PXRE重组质粒,并与人孕烷X受体表达质粒共转染HepG2细胞,从而建立报告基因模型。运用该模型考察了9种常见中药对人孕烷X受体的激活作用,即对UGT1A1的潜在诱导作用。结果将UGT1A1启动子片段克隆到pGL3载体上,构建了pGL3-PXRE重组质粒,成功构建了报告基因模型,并考察了多种常见中药的提取物通过人孕烷X受体途径对UGT1A1的诱导作用。结论成功地建立了基于人孕烷X受体的UGT1A1的报告基因模型,为人孕烷X受体激活剂的体外筛选提供了一种有效的方法。
OBJECTIVE: To establish a reporter gene model of UGT1A1 based on human pregnane X receptor (hPXR), and to investigate the potential induction of Chinese herbal extract on UGT1A1 through human pregnane X receptor pathway. METHODS: Human and genomic DNA was used as a template to amplify the distal and proximal promoters of UGT1A1 and ligated into pGL3 vector to construct pGL3-PXRE recombinant plasmid and co-transfected with HepG2 cells with human pregnane X receptor expression plasmid Reporter model. The model was used to investigate the activation of human pregnane X receptors by nine kinds of common Chinese medicines, that is, the potential induction of UGT1A1. Results The UGT1A1 promoter fragment was cloned into pGL3 vector and the recombinant plasmid pGL3-PXRE was constructed. The reporter gene model was successfully constructed and the induction of UGT1A1 by the human pregnane X receptor pathway was examined . Conclusion The reporter gene model of UGT1A1 based on human pregnane X receptor was successfully established, which provided an effective method for the in vitro screening of human pregnane X receptor activator.