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。
Object:
Obesity has emerged as a worldwide health issue, and has close relationship with many diseases, including type2diabetes, hypertension, coronary heart disease, stroke, dysregulation of lipid metabolism, insulin resistance, non-alcoholic liver disease, asthma and arthritis, etc. The much more fat accumulation in adipocytes is the core factor in occurrence of development of obesity. In1997, myostatin was found by American scientists, which could inhibit the growth of skeletal muscles. So far, the skeletal muscles were considered as one of the most important issues that consume the energy, and without the function of inhibiting muscles growth by myostatin may increase the energy consumption in muscles. The pathway probably indicates a new direction to study the pathogenesis of obesity. In this study, we constructed pcDNA3.1(+)-mMSTN vector containing murine MSTN full coding sequence. We will observe the influence of MSTN overexpression on the weight, epididymal fat, glucose and lipid metabolism in obesity mices in the future. Furthermore we will compare the relationship between MSTN level and obesity.
Methods:
1The construction of pcDNA3.1(+)-mMSTN plasmid containing murine MSTN full coding sequence:Total RNA from BALB/c mice were extracted from murine liver, transcribed reversly into cDNA, then amplified and gained high copies of murine MSTN cDNA full sequences. The pcDNA3.1(+) plasmid were performed to high copies. BamH I、 Apa I were used to cut the plasmid to have the vector fragments. Then we ligated the murine MSTN cDNA sequences and the vector fragments by T4lignase and constructed the pcDNA3.1(+)-mMSTN plasmid containing murine MSTN coding sequence successfully.
2The identification of pcDNA3.1(+)-mMSTN plasmid containing murine MSTN coding sequence in vitro:The plasmid was transfected into murine3T3-L1cells by liposome transfection method and gathered the RNA from the cells after48hours, then observed MSTN expression of cDNA by real-time PCR method.
Results:
1Murine MSTN coding sequence was amplified successfully and ligated with vector fragments by T4ligase. The construction of pcDNA3.1(+)-mMSTN plasmid containing murine MSTN coding sequence was succeed.
2pcDNA3.1(+)-mMSTN plasmid was transfected into murine3T3-LI cells by liposome transfection method. It was confirmed that pcDNA3.1(+)-mMSTN plasmid could well expressed in murine3T3-L1adipocytes..
Conclusion:
1mMSTN expression plasmid pcDNA3.1(+)-mMSTN was constructed successfully and could express murine MSTN protein in vitro murine preadipocytes3T3-L3cells and in vivo mices. This is a convenient tool for MSTN study.
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