NPC1L1基因单核苷酸多态性分析及其与胆固醇结石病发生相关机制的研究

Niemann Pick C1 like 1 (NPC1L1) locates at canalicular membrane of hepatocytes in human. It is responsible for the re-uptake of biliary cholesterol and thus, can regulate the cholesterol content in bile. We found that in gallstone patients, the expression of NPC1L1 was lower and g1679 single nucleotide polymorphism (SNP) was associated with gallstone disease. However, details analysis of all the SNPs in the whole sequence of this gene and their relation with gallstone disease has not been performed yet. .This project will analyze all the SNPs in the promoter and coding sequence of NPC1L1 gene and their relation with gallstone patients from general population, gallstone pedigrees and newly diagnosed gallstone patients from population for gallstone-prediction so as to reveal the gallstone-related specific genotypes and haplotypes. We will also study the effects of SNPs on the difference of gene transcription and translation, intracellular and membrane protein expression, the ratio of cholesterol absorption and the effect on the biliary cholesterol content, which may be the reason leading to different susceptibility of gallstone formation between individuals. These specific genotypes and haplotypes are of key value in prediction of subjects highly susceptible to gallstone disease.

尼曼匹克C1样1蛋白(NPC1L1)在人肝细胞胆小管侧膜表达，通过摄取胆汁中胆固醇，调节胆汁胆固醇饱和度。我们发现胆石病人肝脏NPC1L1表达降低，且NPC1L1基因g1679位点单核苷酸多态性(SNP)与胆石病相关，但对该基因全序列的SNP与胆石病的关系及其机制，目前尚未有完整的研究报道。本课题将分析该基因启动子和编码区域所有SNP位点，以一般胆石病人群、胆石病家系以及胆石病预测基地中新发胆石病人为对象，揭示与胆石病相关的特征性基因型和单倍型。进一步从体外细胞和体内动物模型，研究不同SNP位点在基因转录、翻译、蛋白在胞浆和胞膜的表达差异以及因此导致的对胆固醇摄取率影响和对胆汁胆固醇含量的调节，从功能上明确NPC1L1基因SNP通过影响肝细胞胆固醇摄取以及胆汁胆固醇饱和度，是导致个体对胆石病易感性差异的重要机制。揭示与胆石病相关的NPC1L1基因型和单倍型对筛选胆石病高危人群具有重要意义。

本课题研究了NPC1L1基因启动子及外显子单核苷酸多态性位点，并发现rs2073548多态性与胆石病具有相关性MAF: OR=1.17，95%CI:0.98-1.39，rs2072183多态性MAF: OR=1.30；95%CI：1.09-1.55，P<0.01，并且与胆石病相关的单倍型2-2，OR=1.23，95%CI：1.029-1.470，P=0.02。肝脏NPC1L1的mRNA表达与g1679C>G基因型不同个体其表达存在差异。NPC1L1启动子活性受到胆固醇含量调节，与对照比较，胆固醇(40 μg/ml) 或依泽麦布(15 μM/ml) 均显著抑制NPC1L1启动子活性(P<0.05)。相反，辛伐他汀(40 μM/ml)增加NPC1L1启动子活性(P<0.05)。但含不同多态性序列的启动子间其转录活性没有显著差异。经不同浓度Ezetimibe（20、30、60μM）处理抑制NPC1L1后，且随着浓度升高，新生的自噬体增多，提示胆固醇的水平下降，增强细胞内自噬。检测例胆石病人和例对照病人检测脂肪组织OCP发现DDE和HCH含量显著增高；OCP增高者，肝脏胆固醇转运蛋白ABCG5/G8的表达显著增加，并且脂肪酸合成途径激活，表现为关键酶FAS，SCD1的mRNA表达增加。采用靶向代谢组学发现，OCP增高组病人，肝脏脂肪酸含量增加。且在细胞水平进一步证实了OCP具有上调肝细胞脂肪酸合成的作用。采用荟萃分析方法进一步评价了多个胆石病易感基因，并显示胆固醇7alpha-羟化酶启动子A-204C多态性位点与胆石病相关性(OR = 1.05, 95% CI: 0.91 − 1.22, P = 0.48)；ABCG8基因多态性位点与胆石病相关性分别为：D19H (OR = 2.43, 95%CI: 2.23–2.64, P<0.001); Y54C (OR = 1.36, 95%CI: 1.01–1.83, P = 0.044)。通过多因素回归分析，对NHANES人群数据中环境因子与人体代谢参数分析，结果发现：尿液mandelic acid以及strontium含量与血清睾酮水平具有负相关性OR=2.12 (95% CI: 1.07,4.21; P for trend = 0.044)以及OR=1.84 (95% CI: 1.02, 3.34; P for trend =

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