兔MODY-2模型的构建及药物筛选

发布时间：2018-05-01 00:06

  本文选题：MODY2 + CRISPR/Cas9　； 参考：《吉林大学》2017年硕士论文

【摘要】：糖尿病(Diabetes mellitus,DM)是一种代谢性疾病,由遗传因素与环境因素共同作用引起,特点是高血糖,胰岛素抵抗和体内胰岛素不足。糖尿病的危险因素包括基因突变、家族遗传史、体型肥胖、饮食应激等。糖尿病主要分为以下四大类:⑴:1型糖尿病,胰岛素依赖型糖尿病,由体内胰岛素绝对缺乏引起;⑵:2型糖尿病,成人起病型糖尿病,患者体内产生胰岛素的能力并没有完全丧失,有的甚至产生过多,但是胰岛素的作用效果较差;⑶:特殊型糖尿病,由环境因素和遗传因素引起的,病因相对明确;⑷:妊娠糖尿病,妊娠期间才被诊断出的糖尿病。成人起病的青少年糖尿病(MODY)在上世纪60年代由Fajns发现,现己明确,MODY具有家族遗传史,青少年开始发病,非肥胖等特征。在欧洲国家,MODY的发病率约占总糖尿病人1%-2%。然而由于MODY患者的独有特征,会出现与1型和2型重叠的现象,所以会发生误诊现象,导致实际患病率被低估。资料统计显示,在英国就有80%以上的MODY患者被误诊,然而MODY的最终确诊需要13年。我国对于MODY的研究起步相对较晚,研究结果表明,MODY占中国人早发或多发糖尿病人家系的2%-10%。虽然我国存在MODY患者诊断困难,而且发病率低的现象。但是研究该病意义重大。第一:正确诊断MODY患者,对病人的治疗有指导意义,研究表明,GCK突变发生的MODY2是最常见的MODY亚型;第二,正确的诊断有助于后期的治疗,因为MODY患者通过饮食和运动即可控制血糖水平,无疑会使病人的心理负担极大地减轻。第三,作为一类显性遗传疾病,在分子层面的正确诊断,能为人们提供遗传类疾病的参考;最后,MODY模型的建立,可以作为疾病研究的模型,有助于疾病发病机理的研究和临床药物的筛选。现已明确GCK是MODY2疾病的致病基因,GCK主要在肝细胞和胰岛β细胞中表达。GCK基因位于染色体7p15~p13,全长约27kb,共有12个外显子。GCK由448个氨基酸残基构成,折叠成大小不等的2个结构域。随着人类对疾病认识逐渐深入,研究发现绝大多数疾病的发生都和基因有关。因此,是否能更有针对性的建立与人类疾病有关的动物模型,选择出针对不同疾病的靶向药物是最具有前沿性的课题。本实验通过CRISPR/Cas9基因敲除技术对GCK基因进行修饰,成功构建GCK基因打靶载体,利用Cas9/sg RNA m RNA注射法获得GCK基因敲除兔。研究结果表明,与正常兔相比,GCK基因敲除兔具有血糖长期升高,糖耐量受损,胰岛素分泌减少等典型的MODY2临床症状。而且,我们对多种临床药物进行了筛选,选取格列美脲,瑞格列奈,西格列汀,二甲双胍和胰岛素这五种临床常用药物进行治疗。针对药物的不同疗效,我们选择格列美脲进行长期给药,通过血清学和病理组织学分析,结果显示MODY2疾病症状发生了明显改善。综上所述,本研究利用CRISPR/Cas9技术,成功构建了GCK基因敲除兔,其可为MODY2临床预防治疗提供可靠的模型,并且筛选出针对MODY2有效药物。
[Abstract]:Diabetes mellitus (DM) is a metabolic disease caused by genetic and environmental factors, characterized by hyperglycemia, insulin resistance and inadequacy of insulin in the body. The risk factors for diabetes include gene mutation, family genetic history, obesity and dietary stress. Diabetes is divided into four major categories: (1): type 1 Diabetes, insulin dependent diabetes, caused by the absolute lack of insulin in the body; (2) type 2 diabetes, adult onset diabetes, the patient's ability to produce insulin is not completely lost, some even produce too much, but the effect of insulin is poor; 3. Special diabetes, caused by environmental and genetic factors The cause of the disease is relatively clear; 4: gestational diabetes, the diagnosis of diabetes during pregnancy. Adult onset adolescent diabetes (MODY) was found by Fajns in the 60s of last century. It is clear that MODY has a family history of heredity, juvenile onset, non obesity, and so on. In European countries, the incidence of MODY is about the total 1%-2%. of the total diabetic patients. And because of the unique features of MODY patients, there will be a phenomenon that overlaps with type 1 and type 2, so the misdiagnosis will occur and the actual prevalence rate is underestimated. Data statistics show that more than 80% of the MODY patients in the UK are misdiagnosed, but the final diagnosis of MODY needs 13 years. Our research on MODY is relatively late, and the results show that MODY accounts for the 2%-10%. of early or multiple diabetic families in China, although the diagnosis of MODY patients is difficult and the incidence is low. However, it is of great significance to study the disease. First, the correct diagnosis of MODY patients is of guiding significance to the treatment of the patients. The study shows that the MODY2 of GCK mutation is the most common MODY subtype; second, correct The diagnosis helps the later treatment, because the MODY patients can control the blood sugar level by diet and exercise, and will undoubtedly reduce the patient's psychological burden. Third, as a class of dominant genetic diseases, the correct diagnosis at the molecular level can provide people with the reference of genetic disease. Finally, the establishment of the MODY model can be used as a disease. The model of disease research helps to study the pathogenesis of disease and screening of clinical drugs. It is now clear that GCK is the pathogenetic gene of MODY2 disease. GCK is mainly expressed in the hepatocytes and islet beta cells in chromosome 7p15~p13, with a total length of about 27KB, and a total of 12 exons.GCK consists of 448 amino acid residues, which fold into different sizes. 2 domains. As human cognition of disease progressively deepened, the study found that most of the diseases were related to genes. Therefore, it is the most advanced topic to choose the target drug for different diseases. This experiment is based on CRISPR/Cas9 gene knockout. In addition to the modification of the GCK gene, the GCK gene targeting vector was successfully constructed and the GCK gene knockout rabbit was obtained by Cas9/sg RNA m RNA injection. The results showed that, compared with the normal rabbit, the GCK gene knockout rabbits had the typical MODY2 clinical symptoms, such as the long rise in blood sugar, the impaired glucose tolerance, the decrease of insulin secretion and so on. Moreover, we were on a variety of clinical symptoms. The bed drugs were screened and selected for the treatment of five common clinical drugs, glimepiride, reaglinide, Sig Leo Dean, metformin and insulin. We chose Glimepiride for a long period of Administration for different therapeutic effects. The results showed that the symptoms of MODY2 were obviously improved. In this study, the GCK gene knockout rabbit was successfully constructed by using CRISPR/Cas9 technology, which could provide a reliable model for the clinical prevention and treatment of MODY2 and screen out effective drugs for MODY2.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R-332

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