二恶英通过芳香烃受体（AhR）调控ABC转运蛋白ABCC2基因的研究

Dioxins can disturb liver bilirubin formation, conversion and metabolism processes, which finally may lead to jaundice. The effects of dioxin on bilirubin formation and conversion have been reported, but the mechanism of bilirubin transport in response to dioxins remains to be elucidated. ABCC2 (ATP-binding cassette sub-family C member 2) gene, generally used in the clinical diagnosis of Dubin-Johnson syndrome, plays an important role in the liver bilirubin secretion process. Using transcriptome analysis, we found that ABCC2 gene was upregulated in mouse hepatoma cells treated by dioxins, and the dioxins affect cellular functions by activation of aryl hydrocarbon receptor (AhR) and AhR-dependent signaling pathway to regulate the transcription and expression of its downstream responsive genes. Therefore, the the aim of this study is to find out the role of ABCC2 in bilirubin metabolism induced by dioxins and to reveal if the observed effects are AhR-dependent. Both of cell and animal models will be employed to verify the ABCC2 expressions at gene and protein expression levels in response to dioxins. By means of molecular methods and AhR knockout animal models, the regulation of AhR on ABCC2 gene expression and bilirubin transport function would be clarified. The study would clarify the role of ABCC2 gene in dioxin-induced liver toxicity. We expected that the study could enrich the understanding of the molecular mechanism of dioxin toxicity, and provide useful experiences for subsequent molecular toxicology.

二恶英可影响肝脏胆红素的形成、转化和代谢等过程，从而可能导致黄疸。二恶英对胆红素形成和转化的分子机理研究的比较清楚，但是对胆红素运输代谢的作用机理还没有报道。ABCC2基因在肝脏分泌胆红素过程中发挥重要作用，在临床上用来诊断鉴别慢性特发性黄疸。利用转录组分析，我们发现ABCC2基因在小鼠肝癌细胞中受二恶英诱导后表达上调，而且二恶英毒性主要是通过芳香烃受体（AhR）调控基因表达而起作用。因此，目前亟待解决的关键问题，即ABCC2基因在二恶英影响胆红素代谢过程中所扮演的角色以及是否受芳香烃受体（AhR）的调控。本项目拟通过对ABCC2基因和蛋白水平表达的研究，明确其对二恶英的响应。通过分子手段和转基因动物模型，以明确AhR对ABCC2基因表达和胆红素运输功能的调控。本项目将进一步阐明ABCC2在二恶英对肝脏毒理过程中的功能，丰富对二恶英毒理并且致病的分子基础及作用机制的认识。

二恶英可影响肝脏胆红素的形成、转化和代谢等过程，从而可能导致黄疸。二恶英对胆红素形成和转化的分子机理研究的比较清楚，但是对胆红素运输代谢的作用机理还没有报道。MRP2(Multidrug resistance protein 2)在肝脏分泌胆红素过程中发挥重要作用。利用转录组分析，我们发现MRP3基因在人肝癌细胞中受二恶英诱导后表达上调，而且二恶英毒性主要是通过芳香烃受体（AhR，aryl hydrocarbon receptor）调控基因表达而起作用。因此，目前亟待解决的关键问题，即MRP2基因在二恶英影响胆红素代谢过程中所扮演的角色以及是否受芳香烃受体（AhR）的调控。我们发现ABCC2基因在小鼠肝癌细胞中受二恶英诱导后，表达上调。利用生物信息学软件，选取人和小鼠ABCC2基因转录起始位点上游3700个碱基和下游299个碱基作为启动子分析的区域，我们发现人和小鼠同源基因ABCC2中有保守的芳香烃受体（AhR）结合的二恶英响应元件，这也说明此位点很有可能就是发挥功能的位点。 利用qRT-PCR技术，TCDD处人肝癌细胞24小时后，我们发现ABCC2基因的表达上调。用AhR的拮抗剂CH223191来共同处理细胞后，ABCC2的表达恢复到对照的水平，说明ABCC2基因的表达受AhR的调控。 利用报告基因质粒（pGL3-basic）来研究ABCC2基因上不同二恶英响应元件的转录活性，初步验证有功能的位点。