利用体细胞突变方法鉴定胆固醇代谢调控基因-生物学 生物物理学专业论文.docxVIP

兰州人学硕士学位论文蒋威摘要 兰州人学硕士学位论文蒋威 摘要 胆固醇是哺乳动物体内最重要的一类甾醇小分子。如果其代谢异常会导致许 多疾病的发生，如体内胆固醇水平过高可引起动脉粥样硬化并进而引发中风以及 冠心病等；另外，胆固醇代谢失调也与老年痴呆症、肥胖、糖尿病等存在密切关 系。生理条件下胆固醇的合成被一套严格且精巧的调控机制所调控，其最主要的 反馈调控途径有两条。第一条是对胆固醇内源合成途径中的限速酶．羟甲基戊二 酰辅酶A还原酶(HMG—CoA Reductase，简称HMGCR)的调控；另外一条是转 录因子SREBP剪接的调控途径。 为了进一步阐明胆固醇内源合成途径调控的分子机制，我们以基于慢病毒的 VBIM(Validation·Based Insertional Mutagenesis)插入突变系统作为诱变工具，结 合体细胞遗传学来寻找胆固醇合成途径中新的调控因子。我们的研究结臬表明， 通过新的诱变方法，筛选得到了多种胆固醇合成调控异常的突变细胞株；鉴定出 一些新型突变体；还得到一些可能是未知基因的克隆，目前这些突变体正在鉴定 过程中。 关键词：胆固醇、胆固醇合成、遗传筛选、插入突变、体细胞遗传学、VBIM、 HMGCR 3 兰州人学硕上学位论文蒋威Abstract 兰州人学硕上学位论文蒋威 Abstract Cholesterol is the principal sterol and it is an essential component of animal cell membranes．Abnormal cholesterol level leads to lots of diseases，such as atherosclerosis，which is the primary cause of heart disease and stroke；Additionally, Alzheimer’S disease，obesity and diabetes are also thought to be related to cholesterol metabolism．Under normal conditions，the level of cholesterol is tightly controlled by a series of feedback systems．There are two known principal feedback pathways in cholesterol homeostasis．The first one is that the activity of HMG．CoA reductase (HMGCR)，which is believed to be the rate—limiting enzyme in cholesterol biosynthesis，is regulated by sterol and nonsterol end—products through multiple mechanisms，among which the sterols-induced degradation of HMGCR is the most important one；another pathway involves the SREBP cleavage． To further study the molecular mechanism in cholesterol biosynthesis，we introduce lentivirus—based VBIM(Validation-Based_Insertional Mutagenesis)insertional mutagenesis system ills tools，and incorporate with somatic cell genetics to screen the novel regulators involved in cholesterol homeostasis．The results showed that we obtained several kinds of mutant cells that are deficient in cholester01 feedback regulation．Additionally,we also get some clones among which one or more new genes may be abnormal，which are being identified currently． Keywords：Cholesterol，Choleste