小鼠Myostatin基因表达载体的构建及体外表达鉴定

发布时间：2018-01-02 14:11

本文关键词：小鼠Myostatin基因表达载体的构建及体外表达鉴定　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

【摘要】：目的：随着现代生活习惯的改变和生活质量的提高,很多人开始胖起来,肥胖症已渐渐成为危害全球人类健康的重要疾病,与2型糖尿病、高血压、心脑血管疾病、脂代谢异常、胰岛素抵抗、非酒精性肝病、哮喘以及关节炎等等多种疾病的发生发展密切相关。脂肪细胞中脂质的过度积聚是肥胖症形成其中的中心环节,多种因素参与过程其中,但是发病机制还不完全清楚,因此关于此方面的研究,对肥胖症的防治有重要意义并逐渐成为热门的课题。1997年美国基因学家发现肌肉生长抑素(Myostatin)及其基因(MSTN),对肌肉生长具有抑制作用,目前研究者普遍认为肌肉是全身最大的耗能组织之一,肌肉生长抑素的缺乏可导致肌肉异常增生,同时可能出现肌肉耗能增加,从而调控能量的分配,对认识肥胖的发病机制可能有重大的意义。本研究拟用质粒构建技术及基因表达技术,构建该基因的表达载体,期待用于观察MSTN基因过表达对肥胖小鼠体重、内脏脂肪含量、糖脂代谢等的影响,进而进一步通过该工具观察探讨MSTN与肥胖症的关系。方法： 1小鼠MSTN蛋白表达质粒pcDNA3.1(+)-mMSTN的构建： 1.1mMSTN cDNA插入片段的扩增：从ICR小鼠肌肉组织中提取总RNA,经RT-PCR,获得大量拷贝的MSTN cDNA,然后插入pEASY-T1克隆载体中,经BamHI、ApaI双酶切后,获得带有酶切位点的小鼠MSTN表达序列片段。 1.2载体片段的获得：以pcDNA3.1(+)质粒转化TOP10感受态细菌,得到该质粒的大量拷贝后,大量提取,经BamH I、Apa I双酶切后,进行琼脂糖电泳回收,获得用于构建mMSTN表达质粒的载体片段。 1.3应用T4DNA连接酶,将带有酶切位点的mMSTN扩增片段,与pcDNA3.1(+)质粒酶切以后的载体片段进行连接,构建成pcDNA3.1(+)-mMSTN质粒。 2pcDNA3.1(-)-mZAG表达质粒的体外鉴定：应用阳离子脂质体转染方法将pcDNA3.1(+)-mMSTN质粒瞬时转染到小鼠3T3-L1前脂肪细胞中,48h后收集细胞提起RNA,逆转录合成cDNA后,用real-time PCR方法观察细胞中MSTN的表达情况。结果： 1.成功克隆小鼠MSTN cDNA全序列,并将其与pcDNA3.1(+)载体片段连接,构建成pcDNA3.1(+)-mMSTN表达质粒。 2.在体外培养的小鼠3T3-L1前脂肪细胞系中,证实MSTN表达质粒pcDNA3.1(+)-mMSTN可以在脂肪细胞中良好表达。结论： 1成功构建了pcDNA3.1(+)-mMSTN表达质粒,在体外能良好表达鼠源MSTN蛋白,是研究MSTN作用的一种有效工具。
[Abstract]:Objective: With the change of modern life habits and the improvement of quality of life, many people begin to gain weight. Obesity has gradually become an important disease that endangers the global human health, and type 2 diabetes mellitus, hypertension, cardiovascular and cerebrovascular diseases. Abnormal lipid metabolism, insulin resistance, non-alcoholic liver disease, asthma, arthritis and other diseases are closely related to the occurrence and development of many diseases. The excessive accumulation of lipid in adipocytes is the central link in the formation of obesity. A variety of factors participate in the process, but the pathogenesis is not completely clear, so the research on this aspect. In 1997, American geneticists discovered myostatin (Myostatin) and its gene (MSTN). At present, it is widely believed that muscle is one of the largest energy-consuming tissues in the whole body, and the deficiency of muscle somatostatin can lead to abnormal proliferation of muscle and increase of muscle energy consumption. The regulation of energy distribution may be of great significance in understanding the pathogenesis of obesity. This study intends to use plasmid construction technology and gene expression technology to construct the expression vector of the gene. It is expected to be used to observe the effects of MSTN gene overexpression on body weight, visceral fat content, glucose and lipid metabolism in obese mice, and to further explore the relationship between MSTN and obesity through this tool. Methods: 1 Construction of mouse MSTN protein expression plasmid pcDNA3.1 (mMSTN): Amplification of 1.1 mMSTN cDNA insert fragment: total RNAs were extracted from muscle tissues of ICR mice and a large number of copies of MSTN cDNA were obtained by RT-PCR. Then inserted into the pEASY-T1 clone vector and digested with BamHIHI-ApaI, the MSTN expression sequence of mice with restriction site was obtained. 1.2 the vector fragment was obtained: the plasmid pcDNA3.1 () was transformed into TOP10 receptive bacteria. After a large number of copies of the plasmid were obtained, a large number of copies were extracted and BamH I was used. After Apa I double enzyme digestion, agarose electrophoresis was used to recover the vector fragment used to construct mMSTN expression plasmid. 1.3 using T4 DNA ligase, the mMSTN fragment with restriction site was amplified and ligated with the vector fragment of pcDNA3.1 () plasmids. PcDNA3.1 (mMSTN) plasmid was constructed. In vitro identification of pcDNA3.1mZAG expression plasmid: cationic liposome transfection of pcDNA3.1 (). Mouse 3T3-L1 preadipocytes were transiently transfected with -mMSTN plasmid. After 48 hours, the cells were collected and cDNA was synthesized by reverse transcription. The expression of MSTN in the cells was observed by real-time PCR method. Results: 1. The whole mouse MSTN cDNA sequence was cloned and ligated with pcDNA3.1 () vector fragment to construct pcDNA3.1. MMSTN expression plasmid. 2. In the mouse 3T3-L1 preadipose cell line cultured in vitro, it was confirmed that the MSTN expression plasmid pcDNA3.1 (mMSTN) could be well expressed in adipocytes. Conclusion: 1. PcDNA3.1 (mMSTN) expression plasmid was successfully constructed, which can express murine MSTN protein well in vitro. It is an effective tool to study the role of MSTN.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R589.2;R3416

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