优化的肝富集转录因子和异源物核受体组合协同调控肝细胞尿素循环和药物代谢的研究

Urea cycle, one of two ammonia metabolism pathways in liver cells, is more important than glutamine synthesis pathway. Our previous study confirmed that the recovery of urea cycle can increase the ability of ammonia tolerance and reduction of HepG2 cells, but did not prolong the survival time of rat with liver failure by treatment of bioartificial liver (BAL) system. The main reason is that various hepatotoxic toxins or drugs cannot be effectively degraded due to the low expression of variety of cytochrome P450 (CYP) in HepG2 cells. In this study, on the basis of bioinformatics analysis and by constructing the lentiviral expression vectors, which contain the nucleotide sequences of human liver enriched transcription factor (LETF) or xenobiotic nuclear receptor that binds the core promoter or enhancer region of urea cycle key enzymes and several CYPs genes, we will select an optimum combination of LETFs plus xenobiotic nuclear receptors that can make a significant improvement for urea cycle in ammonia metabolism and drug detoxification. Furthermore, we will use this comination to establish recombinant HepG2 cell lines, investigate their molecular mechanism about the effects of this combination on ammonia metabolism and drug detoxification by promoter analysis, gene chip and signal transduction analysis, as well as evaluate their practical value through the drug metabolism experiments in vitro and treatments of rat models with hepatic failure using a micro-BAL system loaded with the recombinant HepG2 cells. The implementation of the project is expected to establish human derived hepatocytes that can be conveniently used for BAL and detection of drug hepatotoxicity.

肝细胞氨代谢两条通路中尿素循环更为重要。前期我们研究证实尿素循环的恢复虽然能提高HepG2细胞耐氨和降氨能力，但应用于生物人工肝（BAL）系统并不能延长肝衰大鼠的的生存时间，主要原因是HepG2细胞的多种细胞色素P450（CYP）表达低下，各种肝毒性毒素（药物）无法有效降解。本研究在生物信息学分析的基础上，通过构建含肝细胞尿素代谢关键酶和几种主要CYP基因核心启动子和增强子区域共有的肝富集转录因子（LETF）或异源物核受体的慢病毒表达载体，筛选对肝细胞氨代谢（尿素循环通路）和药物代谢均有重要影响的最优LETF+异源物核受体组合，并以此建立重组HepG2细胞。从启动子分析、基因芯片和信号传导等角度探明这种组合对HepG2细胞氨代谢和药物解毒等影响的分子机制，并通过体外药物代谢实验和BAL治疗肝衰大鼠模型实验，评价其实际应用价值。有望建立能同时用于BAL和药物肝毒性检测的取用便捷的人源肝细胞。

一、本研究利用生物信息学结合实验技术分别对肝细胞氨代谢尿素通路的精氨酸酶1（Arg1）、鸟氨酸转氨甲酰酶（OTC）和氨基甲酰磷酸合成酶1（CPS1）三种基因启动子进行分析，发现：1）Arg1启动子的-160nt ~ +28nt区域保留了最大活性，该区域的C/EBPβ结合位点对Arg1活性影响最大。2）OTC启动子-165nt ~ -90nt区域存在两个HNF4α结合位点，PGC1α可明显增强HNF4α对OTC启动子的转录激活作用，PGC1α与HNF4α结合于OTC启动子相同位置。3）CPS1启动子的-70nt ~ +73nt存在重要的HNF3β结合位点，CPS1启动子的活性随着HNF3β过表达而增加；不同肝细胞株在转染HNF3β后，随着NH4Cl浓度的升高尿素含量均有不同程度的增加。二、基于以上的结果并结合文献资料，本研究构建多种肝富集转录因子（LETF）+异源物核受体的腺病毒组合，优选出“C/EBPβ+HNF3β+HNF4α”、“C/EBPβ+HNF3β+HNF1α”和“C/EBPβ+HNF4α+HNF6” 三种组合能有效促进尿素循环5种关键酶的表达，随后再与孕烷素核受体（PXR）和组成性雄烷受体（CAR）进行重新组合，最终优选出 “C/EBPβ+HNF4α+HNF6+PXR”和“C/EBPβ+HNF4α+HNF6+CAR”两种组合能有效促进尿素循环5种关键酶和五种CYP酶（CYP1A1、CYP2B6、CYP2C9、CYP2D6和CYP3A4）的表达，并成功构建稳定过表达两种优化组合的重组肝细胞。三、本研究还发现氨诱导后肝细胞的自噬水平可明显上升，敲低PXR（PXR-）的细胞株则强化这种现象，利福平（PXR激活剂）也有类似的作用。机制分析发现能量感应分子p-AMPKβ1水平在PXR组中明显高于野生型细胞株对照、P53可直接结合于AMPKβ1启动子（-253nt ~ -19nt）区域、PXR与P53之间存在蛋白相互作用。.科学意义：1）C/EBPβ+HNF4α+HNF6+PXR和C/EBPβ+HNF4α+HNF6+CAR重组慢病毒组合均对利于人肝细胞尿素循环和药物代谢功能的提升，且前者效果更好，基于该组合构建的人肝细胞株有望为进一步的实验性应用提供取用便捷的肝细胞来源。2）PXR是氨诱导的肝细胞自噬形成的干扰因素，可为临床PXR激活剂药物引发的肝毒性提供新的解释。