本文選題：MODY2 + CRISPR/Cas9　； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：糖尿病(Diabetes mellitus,DM)是一種代謝性疾病,由遺傳因素與環(huán)境因素共同作用引起,特點(diǎn)是高血糖,胰島素抵抗和體內(nèi)胰島素不足。糖尿病的危險(xiǎn)因素包括基因突變、家族遺傳史、體型肥胖、飲食應(yīng)激等。糖尿病主要分為以下四大類(lèi):⑴:1型糖尿病,胰島素依賴(lài)型糖尿病,由體內(nèi)胰島素絕對(duì)缺乏引起;⑵:2型糖尿病,成人起病型糖尿病,患者體內(nèi)產(chǎn)生胰島素的能力并沒(méi)有完全喪失,有的甚至產(chǎn)生過(guò)多,但是胰島素的作用效果較差;⑶:特殊型糖尿病,由環(huán)境因素和遺傳因素引起的,病因相對(duì)明確;⑷:妊娠糖尿病,妊娠期間才被診斷出的糖尿病。成人起病的青少年糖尿病(MODY)在上世紀(jì)60年代由Fajns發(fā)現(xiàn),現(xiàn)己明確,MODY具有家族遺傳史,青少年開(kāi)始發(fā)病,非肥胖等特征。在歐洲國(guó)家,MODY的發(fā)病率約占總糖尿病人1%-2%。然而由于MODY患者的獨(dú)有特征,會(huì)出現(xiàn)與1型和2型重疊的現(xiàn)象,所以會(huì)發(fā)生誤診現(xiàn)象,導(dǎo)致實(shí)際患病率被低估。資料統(tǒng)計(jì)顯示,在英國(guó)就有80%以上的MODY患者被誤診,然而MODY的最終確診需要13年。我國(guó)對(duì)于MODY的研究起步相對(duì)較晚,研究結(jié)果表明,MODY占中國(guó)人早發(fā)或多發(fā)糖尿病人家系的2%-10%。雖然我國(guó)存在MODY患者診斷困難,而且發(fā)病率低的現(xiàn)象。但是研究該病意義重大。第一:正確診斷MODY患者,對(duì)病人的治療有指導(dǎo)意義,研究表明,GCK突變發(fā)生的MODY2是最常見(jiàn)的MODY亞型;第二,正確的診斷有助于后期的治療,因?yàn)镸ODY患者通過(guò)飲食和運(yùn)動(dòng)即可控制血糖水平,無(wú)疑會(huì)使病人的心理負(fù)擔(dān)極大地減輕。第三,作為一類(lèi)顯性遺傳疾病,在分子層面的正確診斷,能為人們提供遺傳類(lèi)疾病的參考;最后,MODY模型的建立,可以作為疾病研究的模型,有助于疾病發(fā)病機(jī)理的研究和臨床藥物的篩選�，F(xiàn)已明確GCK是MODY2疾病的致病基因,GCK主要在肝細(xì)胞和胰島β細(xì)胞中表達(dá)。GCK基因位于染色體7p15~p13,全長(zhǎng)約27kb,共有12個(gè)外顯子。GCK由448個(gè)氨基酸殘基構(gòu)成,折疊成大小不等的2個(gè)結(jié)構(gòu)域。隨著人類(lèi)對(duì)疾病認(rèn)識(shí)逐漸深入,研究發(fā)現(xiàn)絕大多數(shù)疾病的發(fā)生都和基因有關(guān)。因此,是否能更有針對(duì)性的建立與人類(lèi)疾病有關(guān)的動(dòng)物模型,選擇出針對(duì)不同疾病的靶向藥物是最具有前沿性的課題。本實(shí)驗(yàn)通過(guò)CRISPR/Cas9基因敲除技術(shù)對(duì)GCK基因進(jìn)行修飾,成功構(gòu)建GCK基因打靶載體,利用Cas9/sg RNA m RNA注射法獲得GCK基因敲除兔。研究結(jié)果表明,與正常兔相比,GCK基因敲除兔具有血糖長(zhǎng)期升高,糖耐量受損,胰島素分泌減少等典型的MODY2臨床癥狀。而且,我們對(duì)多種臨床藥物進(jìn)行了篩選,選取格列美脲,瑞格列奈,西格列汀,二甲雙胍和胰島素這五種臨床常用藥物進(jìn)行治療。針對(duì)藥物的不同療效,我們選擇格列美脲進(jìn)行長(zhǎng)期給藥,通過(guò)血清學(xué)和病理組織學(xué)分析,結(jié)果顯示MODY2疾病癥狀發(fā)生了明顯改善。綜上所述,本研究利用CRISPR/Cas9技術(shù),成功構(gòu)建了GCK基因敲除兔,其可為MODY2臨床預(yù)防治療提供可靠的模型,并且篩選出針對(duì)MODY2有效藥物。
[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.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R587.1;R-332

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