中国北方多民族人群药物代谢酶CYP450与肿瘤耐药转运蛋白MDR1基因表观遗传多态性及机制的研究

Cytochrome P450 (CYP450) drug metabolic enzymes and drug transportor (P-gp, MDR1) have been accepted to relate tumor multidrug resistance, but the exact epigenetic modulating mechanism is still unknown. This research purpose is to study on the relationship between frequencies of DNA methylation and the single nucleotide polymorphisms (SNPs) of CYP450s and MDR1 in northern mianland Chinses Tibetan, Mongolian, Uyghur and Han multiethnic populations. The methods of matrix assisted laser desorption/ ionization-time of flight - mass spectrum (MALDI-TPF- MS),polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and liquid chromatography tandem mass spectrometric (LC-MS/MS), will be using to detect the epigenetic genotypes, alleles and pharmacokinetics phenotypes in the samples of peripheral blood, hepatocellular carcinoma and paracancerous liver tissue in healthy subjects and hepatocellular caricinoma patients in vivo, and in human hepatoma transgenetic mCAR-HepG2 cells in vitro, respectively. The present research will focus on determinating and comparing the effects of oxidative stress on the gene expression of CYP450 and the upstream transcription factor, for example, constitutive androstane receptor (CAR) promotor region, in CpG island methylation 5-methylcytosine (5-mC) and /or 5-hydroxymethyl cytosine (5-hmC), and on the phenotypes of the antitumor drugs phamacokinetics in clinical cancer patients in vivo and in vitro. The present results will not only be likely to provide the new explain for etiology of tumor multidrug resistance, but also might be expected to elucidate the pharmacoepigenetics modulating mechanisms of cytochrome P450 enzymatic phenotypes by endoplasmic reticular oxidative /nitrosative stress in the course of xenobiotic metabolic process, and then might be discovered more specific and selective drug target for reversing or inhibiting tumor multidrug resistance, provided novel therapeutic theoretics and experimental evidence.

药物氧化代谢酶CYP450及转运蛋白MDR1基因表达与恶性肿瘤多药耐药的发生密切相关，但其确切的表观遗传学修饰机制尚知之甚少。本研究拟在前期工作的基础上，以中国北方及西北地区不同遗传背景及海拔（缺氧）居住环境的多民族健康志愿者(藏族、蒙古族、维吾尔族及汉族)外周血、肝癌患者外周血、癌与癌旁配对组织（炎-癌氧化应激）、人肝癌转基因mCAR-HepG2细胞模型为对象，采用MALDI-TOF-MS、RFLP-PCR、LC-MS等方法，考察CYP450与MDR1基因组中DNA甲基化分布频率与单核苷酸多态性（SNPs）分布频率的异同点；氧化应激对CYP450基因及上游转录因子CAR启动子区CpG岛甲基化或羟甲基化的影响，以及对抗肿瘤药物代谢表型的影响。研究旨在阐明CYP450与MDR1基因的遗传多态性与表观遗传多态性之间的相关性及分子机制，为逆转肿瘤多药耐药个体化治疗提供药物作用新靶点和实验依据。

本课题以中国藏族、蒙古族、维吾尔族及汉族人群为对象，①采用PCR-RFLP法和Sanger测序法，考察了CYP1A2、CYP2B6及iNOS等位基因分布频率，并与已经报道的高加索白种人群、非洲黑种人群基因多态性频率进行了比较和分析。②在中国汉族及维吾尔族人群中发现了10个新的CYP2W1单核苷酸多态性位点（SNPs），结果提示与CYP2W1酶代谢功能及结直肠癌发生易感性相关。③考察了中国藏族、蒙古族、维吾尔族及汉族人群中CYP2D6和eNOS的等位基因频率、单倍体连锁不平衡分布频率；④采用重亚硫酸盐修饰-PCR测序法，对上述四民族人群CYP2D6和eNOS基因启动子区胞嘧啶-鸟嘌呤二核苷酸（CpG）位点甲基化水平进行了定量分析，探讨了基因变异频率与DNA甲基化频率的相关性。⑤采用焦磷酸测序等方法，考察了中国北方汉族人群CYP2C8、CYP2C9、CYP3A5、UGT1A1和ABCB1的SNPs分布频率，及其对口服吡格列酮、咪唑斯汀后药物代谢动力学参数的影响，探讨了基因型-代谢表型相关性及机制。⑥考察了小檗碱（BBR）在雄甾烷核受体（CAR）转基因肝癌细胞内的分布动力学、对CAR及其靶基因CYP2B6与CYP3A4表达、全基因组DNA甲基化、启动子区CpG位点甲基化的影响。⑦采用重组人源CYP450亚型酶蛋白，探讨了抗癫痫新药氯桂丁胺（3.4-DCPB）代谢表型与CYP450酶亚型之间的相互作用及机制。⑧评价了数个抗肿瘤新药或新化合物（GLB、W-1、PU-48）的血药浓度经时变化及药代动力学参数、母药代谢途径鉴定、纳米制剂的生物利用度、组织分布及排泄途径分析、血浆蛋白结合率分析，为药动学机制研究提供了新的视角。课题执行期间发表论文15篇，其中SCI收录源期刊原著论文13篇，中文核心期刊综述论文2篇；培养毕业博士生5人，毕业硕士生3人；后续工作期间仍将有数篇论文发表。