Myostatin干扰质粒构建及在C2C12细胞中效果验证.doc

Myostatin干扰质粒构建及在C2C12细胞中效果验证 　作者：吴欣，邵晨昕，顾芸，丁斐，刘梅 【摘要】 目的：研究Myostatin干扰质粒在小鼠骨骼肌成肌细胞系C2C12细胞中的沉默作用。方法：根据Myostatin mRNA序列，选择3个19nts的靶序列，设计并合成包含各正反义靶序列的互补DNA链以及1个无干扰作用DNA链，退火后插入pSilencer2.1-U6表达载体，构建3个RNAi阳性质粒：M1/pSilencer、M2/pSilencer、M3/pSilencer及一个阴性质粒Mc/ pSilencer并转染C2C12细胞，采用实时荧光定量PCR和Western blot观察RNAi对C2C12细胞中Myostatin mRNA和蛋白表达的下调作用。结果：构建的3个阳性干扰质粒中M1/pSilencer能显著降低C2C12细胞中Myostatin表达量，随着M1/pSilencer干扰质粒剂量的增加，Myostatin表达量逐渐降低。结论：成功构建Myostatin RNAi质粒，该质粒能下调目的基因Myostatin在C2C12细胞中的表达量。 【关键词】 Myostatin；RNA干扰；C2C12 [Abstract] Objective：To evaluate the role of RNA interference (RNAi) in silencing Myostatin expression in mouse C2C12 cells (myoblast). Methods：Three target sequences and a negative sequence were selected according to Myostatin mRNA sequence，the complementary DNA contained both sense and antisense oligonueleotides were designed and synthesized．After annealing, the double strands DNA were ligated to the pSilencer2.1-U6 siRNA expression vector．Three positive RNAi plasmids named M1/pSilencer, M2/pSilencer, M3/pSilencer, and the nagetive plasmid named Mc/pSilencer were transfected into the C2C12 cells, in which the silencing effect on Myostatin expression was investigated by real-time PCR and Western blot. Results：Among the three RNAi positive plasmids, the M1/pSilencer could reduce Myostatin expression significantly. With the dose of plasmid M1/pSilencer increasing, the expression of Myostatin and mRNA decreased. Conclusion：The results indicated that Myostatin RNAi plasmids were obtained, and the RNAi plasmids could reduce Myostatin expression in C2C12 cells. [Key words] Myostatin；RNAi；C2C12 1997年美国Johns Hopkins大学的研究人员从小鼠骨骼肌cDNA文库中克隆出Myostatin。蛋白质同源性比较证明Myostatin是TGF-β超家族的新成员，又称生长分化因子8。在Myostatin敲除的小鼠体内，肌纤维发生广泛的增生和肥大，导致骨骼肌肌肉质量显著增加[1~4]。Myostatin编码序列发生突变的牛也出现肌肉质量显著增加，呈现“双肌现象”[5，6]。由此可见，Myostatin能抑制骨骼肌的生长，是骨骼肌生长的负性调控因子[3，7]。我们以前的研究表明，在坐骨神经缺损后，失神经支配的腓肠肌Myostatin mRNA和蛋白具有特定的表达模式[8，9]，提示Myostatin在失神经性肌萎缩过程中扮演重要角色。为了进一步探讨Myostatin在失神经肌萎缩中的生物学作用，本实验构建小鼠Myostati