【作者】 姚晓敏；

【导师】 林志新； 李伯良；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 酰基辅酶A:胆固醇酰基转移酶(ACAT)是细胞内唯一催化游离胆固醇和脂肪酸合成胆固醇酯的酶,在细胞内胆固醇代谢平衡中起非常重要的作用。目前,在哺乳动物细胞中发现两种基因编码ACAT(ACAT1和ACAT2)。ACAT1几乎在各种组织细胞中均有表达,与细胞胆固醇代谢平衡直接相关;而在肝肠细胞中特异表达的ACAT2则与外源胆固醇的吸收和脂蛋白装配等密切相关。人类动脉粥样硬化、阿尔海默氏病、胆囊结石等一些重要病变,都与ACAT密切相关。因此,ACAT已成为分子筛药的重要靶蛋白。我们实验室与美国Chang TY教授实验室的合作研究,已首先报道了两种人ACAT基因的基因组结构和ACAT1基因的表达调控。在这些基础上,本论文进行两部分工作,第一部分重点围绕人ACAT2基因表达的可变性剪接和组织特异性进行研究,第二小部分是进行猴ACAT1 cDNA 5′-UTR的克隆分析。首先,进行可变性剪接成熟的人ACAT2 mRNAs及其编码的异构体酶活性的分析研究。人ACAT2基因长约18 kb,包含15个外显子,所有的外显子/内含子连接区序列均为经典的GT/AG序列。通过RT-PCR、克隆和DNA测序分析,获得三种人ACAT2 mRNAs。序列比较表明,它们是从同一pre-mRNA通过可变性剪接产生,外显子的缺失并未导致阅读框的改变;它们编码三种人ACAT2产物,即ACAT2a,包含所有的15个外显子,编码522个氨基酸;ACAT2b,包含除外显子4以外的14个外显子,编码502个氨基酸;ACAT2c,包含外显子1-3、6-7、11-15,编码379个氨基酸;其中ACAT2a与文献报道完全一致,ACAT2b与ACAT2c为新发现的2个ACAT2序列。利用克隆的三种大小不同的人ACAT2 ORF cDNAs构建相应的表达质粒,分别转染ACAT缺陷的AC29细胞进行表达研究,结果显示可表达出相应不同大小的ACAT2蛋白产物;同时在人肝肠细胞株中也检测到了同样不同大小的内源表达ACAT2蛋白产物。这些表明,可表达的内源性ACAT2b和ACAT2c蛋白可能是两种ACAT2异构体,而且它们的氨基更多还原

【Abstract】 Acyl-CoA: cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from long-chain fatty acyl coenzyme A and cholesterol. ACAT plays an important role in cellular cholesterol homeostasis. So far, in mammals, two ACAT genes have been identified. The ACAT1 that is found in almost all of the cells and tissues examined is believed to be crucial for maintaining cholesterol homeostasis while ACAT2 that is selectively expressed in liver and small intestine may be the major enzyme that involved in the dietary cholesterol absorption and assembly of apoB-containing lipoprotein. ACATs also play important roles in some serious human diseases including atherosclerosis, Alzheimer’s disease (AD) and gallstone. For these reasons, ACAT has been considered as one of major pharmaceutical targets for developing CE-lowering, anti-atherosclerosis and/or anti-AD drugs. In collaboration with professor Chang TY′s laboratory (Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA), our laboratory (State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China) has first reported the two human ACAT genomic DNAs, and investigated the expressional regulation of human ACAT1 gene. Based on the previous study, this work is mainly focused on the alternative splicing and tissue-specificity of human ACAT2 gene expression, and cloning and analysis of monkey ACAT1 cDNA 5′-UTR.Firstly, we studied the human ACAT2 mRNA variants from alternative splicing更多还原