MOLECULAR BIOLOGY OF BILE ACID SYNTHESIS

(摘自申请者的摘要)胆汁酸是生理剂 脂溶物质的吸收、运输和处置所必需的 维生素、类固醇和外源生物。胆汁酸是由 胆固醇通过两条途径。主要途径是从 限速酶，胆固醇7a-羟基酶，一直是 研究。CYP7A基因受一种复杂的机制调控。一个 在启动子中的抑制子和激活区已经被映射到 含有对胆汁酸、佛波酯和维甲酸有反应的元素 酸。酵母单杂交系统将用于分离DNA结合 分离出的蛋白质。胆汁酸反馈是一种生理机制 它不仅抑制了细胞色素P7A，而且还对细胞周期蛋白有多种影响 其他肝脏基因的表达。胆汁酸代谢失衡 导致营养吸收不良，肝细胞增殖， 人类的胆汁淤积症、肝硬变和结肠癌。聚合酶 基于链式反应的肝细胞mRNAs差异显示 用于识别受胆汁酸影响的差异基因。胆汁 线粒体甾醇27-启动的酸合成途径 羟基酶和27-羟基胆汁醇7a-羟基酶(氧甾醇7a- 羟基酶)是最近发现的。当胆固醇7a- 羟基酶活性在新生动物中表达水平较低，在 肝脏疾病，在缺乏Cyp7a基因的转基因小鼠中，27- 在肝外组织合成的羟胆雌醇可能是 在肝脏中转化为胆汁酸作为一种代偿机制。 这一途径也调节了有效的氧化甾醇的水平。 胆固醇合成和运输的抑制因子。细胞色素P27基因 已经在脑腱黄瘤病中发现了突变，但 细胞色素P27基因的调控尚不清楚。假设两者都是 类固醇27-羟基酶和氧化类固醇-7a-羟基酶可能起作用 在胆汁酸和氧化甾醇合成调节中的重要作用 测试过。CYP27上游序列中的调节区将是 细胞色素P27/荧光素酶报告基因瞬时横切分析定位 基因、DNase I足迹和凝胶迁移率变化分析。氧合甾醇 将提纯7A-羟基酶并克隆其cDNAs以研究其 在氧固醇和胆汁酸合成的调节中的作用。《长河》 本研究项目的学期目标是了解分子 胆汁酸合成的调控机制及胆汁酸的作用机制 人类胆汁酸代谢和胆固醇动态平衡的疾病。

(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.