大鼠血糖调节相关基因的克隆和功能研究

【作者】 朱国志；

【导师】 方福德；

【作者基本信息】 中国协和医科大学 ， 分子生物学及生物化学， 2000， 博士

【摘要】 糖尿病是全球性医学难题，它已成为继癌症和心脑血管疾病之后的第三大疾患，它也是全世界发病率和病死率最高的五种疾病之一。如不加以有效干预，今后30年内，我国糖尿病患病率将达到5.0-10.0％，届时全国糖尿病患者总数将逾一亿。因此，研究糖尿病的病因、发病机理和糖尿病的最佳防治方案已迫在眉睫。糖尿病是一种具有明显遗传倾向的多基因疾病，其发病机制十分复杂，环境因素和遗传因素又交互作用，共同促成了糖尿病的发生。糖尿病在临床上通常分为两大类，即I型糖尿病(胰岛素依赖性，IDDM)和U型糖尿病(非胰岛素依赖性，NIDDM)，后者占所有糖尿病病例90％以上。NIDDM的基因定位策略主要有候选基因法和全基因组扫描法。候选基因法是指选定糖尿病发病过程中与糖代谢及信号传导有关的基因为研究对象，进而研究它们是否与糖尿病发病过程相关联。候选基因法简便易行，是目前乃至今后研究NIDDM的主要方法之一。然而，到目前为止，应用该方法发现的结果较少，推测NIDDM基因很可能包括许多未发现的新基因。为此，必须选用适当的方法来发现新基因。 我们在研究糖尿病的发生机制和防治策略的过程中，提出了一条新思路，即从正常的血糖自动调节的基因途径入手，先分离、克隆与血糖自动调解相关的基因，再研究这些相关基因在糖尿病状况下有何改变，为确定糖尿病相关或易感基因以及寻找防治途径提供依据。通过颈外静脉—右心房导管插管术建立了大鼠血糖自动调节模型，该模型制作简便，重复性好，人为干扰因素少。随后对该模型大鼠的骨骼肌进行的mRNA差异显示分析。 差异显示是一种用来检测哺乳细胞基因表达差异的方法。自从1992年发明该方法以来，至今已有许多条件优化和改进，逐渐发展成一个比较成熟的鉴定两种状态下的差异表达基因的方法。这些改进包括：锚定引物的数量可以从12条减少到4条；应用长引物可在严格的条件下进行PCR扩增；两步PCR法增强实验结果的可靠性。除了综合以上改进外，本文采用先通过狭缝杂交初步筛选上百条的差异条带、再克隆测序、Northern Blot杂交最后确定差异表达基因的策略。该策略由于先狭缝杂交初步筛选，既显著减少克隆测序的工作量和花费，又能保证从最初的上百条差异条带中最终通过Northern Blot杂交鉴定出最有差异表达特征的基因，从而保证实验的质量。另外，我们以整体动物代替通常的培养细胞系作为实验材料，因为细胞在培养状态下不能很好地代表机体正常的生理状态，尤其不能很好地代表由机体多组织协同参与的代谢活动例如糖代谢。 从最初的181条差异条带中，74条被狭缝杂交证实存在差异表达，其中更多还原

【Abstract】 Diabetes is a disease difficult to treat in worldwide. It is the third disease followed cancer and cardiac disease and one of five diseases with the highest incidence and mortality. If we don’t try to treat it, in the following thirty years, diabetes sufferer in China will reach 5.0-10.0% which mean the total patient number will reach one hundred million. It is necessary to do research on etiology, underlying basis and best precautionary method of diabetes as soon as possible. Diabetes is a multigenes related disease with obvious inherited tendency which has extremely complex basis. In clinical, diabetes is divided into two group: type I diabetes (IDDM) and type II diabetes (NIDDM), the latter is 90% of total diabetes patient. There are two main strategies in NIDDM gene identification: candidate gene strategy and whole genome scanning strategy. Candidate gene strategy is to select genes participating in glucose metabolism and signal transduction as candidate genes. This strategy is simple and one of main strategy in NIDDM research now. But, as yet the result discovered by this strategy is less. This leads to speculate that NIDDM genes are possible those genes have not been discovered. For this purpose, it is necessary to discover glucose metabolism related new genes with proper methods.We have provided a new thought in the research of diabetes here: at first to cloning glucose metabolism related gene, secondly to discover how the related new gene participating into glucose metabolism, to provide basis of identify new candidate gene and of exploit new precautionary method.Firstly, we established SD rat glucose auto-regulating model by use of Jugular Vein right atrium intubation. This model is easy to make and has good repetition, has less artificial interference. Then mRNA differential display assay had been done by the use of this model.mRNA differential display is a method to isolate differential expressed genes from mammalian cell. There has been many modifications since this method has been established which made it a mature method. These modifications include: the number of anchor primers can reduce from 12 to 4; longer primers are used to make PCR more stringent; two-step PCR can enhance reliability. Beside we adopted these modifications, we adopted a strategy that first screening hundreds of differentially expressed bands through slot blot, then cloning and sequencing candidate cDNAs, Northern Blot ensure the differentially expressed genes at last. Because of slot blot更多还原