MOLECULAR BIOLOGY OF BILE ACID SYNTHESIS

（改编自申请人的摘要）胆汁酸是生理试剂 所需的吸收，运输和处置脂溶性 维生素类固醇和异生物素 胆汁酸是由 胆固醇通过两条途径。 主要的路径始于 胆固醇7a-羟化酶是胆固醇代谢的限速酶， research. CYP 7A基因受一种复杂的机制调控。 一 启动子中的阻遏物和激活物区域已经被定位为 含有对胆汁酸、佛波醇酯和视黄酸有反应的元素 酸 酵母单杂交系统将用于分离DNA结合 分离的蛋白质 胆汁酸反馈是一种生理机制 它不仅抑制CYP 7A，而且对细胞色素P450的合成也有不同的影响。 其他利耶基因的表达。 胆汁酸代谢失衡 导致营养吸收不良，肝细胞增殖， 胆汁淤积、肝硬化和结肠癌。 聚合酶 基于链式反应的肝细胞中mRNA的差异显示将 用于鉴定受胆汁酸影响的差异基因。 胆汁 酸合成途径起始于线粒体固醇27- 羟化酶和27-羟甾醇7 α-羟化酶（氧化甾醇7 α-羟化酶）。 羟化酶）最近被发现。 当胆固醇7a- 羟化酶活性在新生动物中以低水平表达， 肝脏疾病，在转基因小鼠缺乏CYp 7a基因，27- 在肝外组织中合成的羟基胆固醇可能 在肝脏中转化为胆汁酸作为一种补偿机制。 该途径还调节氧化固醇的水平， 胆固醇合成和转运的抑制因子。 CYP 27基因 已在腱性黄瘤病中鉴定出突变， CYP 27基因的调控是未知的。 假设两者都 甾醇27-羟化酶和氧甾醇-7 α-羟化酶可能起作用 在调节胆汁酸和氧化固醇合成中的重要作用将 测试. CYP 27上游序列中的调控区将被 通过CYP 27/荧光素酶报告基因的瞬时横断测定作图 基因，DNA酶I足迹和凝胶迁移率变动测定。 氧化固醇 将纯化7 α-羟化酶并克隆其cDNA，以研究其在大肠杆菌中的表达。 在氧化固醇和胆汁酸合成的调节中的作用。 长 本研究项目长期目标是了解分子 胆汁酸合成的调节机制和 胆汁酸代谢和胆固醇稳态的人类疾病。

(adapted from applicant's abstract) Bile acids are physiological agents required for the absorption, transport and disposal of lipid-soluble vitamins, steroids and xenobiotics. Bile acids are synthesized from cholesterol by two pathways. The major pathway starting with the rate-limiting enzyme, cholesterol 7a-hydroxylase, has been the focus of research. The gene CYP7A is regulated by a complex mechanism. A repressor and an activator region in the promoter have been mapped to contain elements responsive to bile acid, phorbol esters and retinoic acid. Yeast one-hybrid system will be used to isolate DNA-binding proteins isolated. Bile acid feedback is a physiological mechanism which not only inhibits CYP7A but also has a diverse effect on the expression of other lier genes. Imbalance in bile acid metabolism causes malabsorption of nutrients, proliferation of hepatocytes, cholestasis, liver cirrhosis and colon cancer in humans. Polymerase chain reaction based-differential display of mRNAs in hepatocytes will be used to identify genes differentially affected by bile acid. Bile acid synthesis pathway initiated with mitochondrial sterol 27- hydroxylase, and 27-hydorxycholeserol 7a-hydorxylase (oxysterol 7a- hydroxylase) has been uncovered recently. When cholesterol 7a- hydroxylase activity is expressed at a low level in neonatal animals, in liver disease, and in transgenic mice deficient the CYp7a gene, 27- hydroxycholestrerol synthesized in the extrahepatic tissues may be converted to bile acids in the liver as a compensatory mechanism. This pathway also regulate the levels of oxysterols which are potent repressors of cholesterol synthesis and transport. The CYP27 gene mutations have been identified in cerebrotendinous xanthomatosis but regulation of the CYP27 gene is unknown. The hypothesis that both sterol 27-hydorxylase and oxysterol-7a-hydroxylase may play important roles in regulation of bile acid and oxysterol syntheses will tested. Regulatory regions in the CYP27 upstream sequence will be mapped by transient transection assay of CYP27/luciferase reporter genes, DNase I foot printing and gel mobility shift assays. Oxysterol 7a-hydroxylase will be purified and cDNA will be cloned to study its role in the regulation of oxysterols and bile acid syntheses. The long term objective of this research project is to understand molecular mechanisms of regulation of bile acid synthesis and mechanisms of human diseases in bile acid metabolism and cholesterol homeostasis.