Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass
Myostatin short interfering hairpin RNA gene transfer increases skeletal muscle mass
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DOI：
10.1002/jgm.946
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发表时间：
2006-09-01
影响因子：
3.5
通讯作者：
Gonzalez-Cadavid, Nestor F.
中科院分区：
文献类型：
--
作者：
Magee, Thomas R.;Artaza, Jorge N.;Gonzalez-Cadavid, Nestor F.
背景肌生长抑制素负性调节骨骼肌生长。肌肉生长抑制素敲除小鼠表现出肌肉肥大和减少的间质纤维化。我们研究了是否表达针对肌肉生长抑制素的短发夹干扰RNA（shRNA）质粒和使用电穿孔转导将增加局部骨骼肌mass.Methods短干扰RNA（siRNA）靶向肌肉生长抑制素与肌肉生长抑制素表达质粒共转染到HEK 293细胞中，并通过Western blot鉴定肌肉生长抑制素沉默。将相应的shRNA克隆到质粒shRNA表达载体中。将肌生成抑制素或随机阴性对照shRNA质粒分别注射并电穿孔到9只大鼠的胫骨前肌或其对侧肌肉中，2周后处死。另外6只大鼠接受β-半乳糖苷酶报告质粒，并在第1、2和4周处死。通过β-半乳糖苷酶表达检测质粒的摄取，而通过实时聚合酶链反应（PCR）和Western印迹法测定肌肉生长抑制素的表达。肌纤维大小用组织化学法测定。PAX 7免疫组化法检测卫星细胞增殖情况。结果β-半乳糖苷酶报告质粒在第1周和第2周时在胫骨前肌中有表达，但在第4周时表达减弱。肌肉生长抑制素shRNA使肌肉生长抑制素mRNA和蛋白表达分别降低27%和48%。胫骨前肌重量、纤维尺寸和MHCII分别增加10%、34%和38%。结论这是第一次证明，肌肉生长抑制素shRNA基因转移是一个潜在的策略，以增加肌肉质量。版权所有（c）2006约翰威利父子有限公司。
Background Myostatin negatively regulates skeletal muscle growth. Myostatin knockout mice exhibit muscle hypertrophy and decreased interstitial fibrosis. We investigated whether a plasmid expressing a short hairpin interfering RNA (shRNA) against myostatin and transduced using electroporation would increase local skeletal muscle mass.Methods Short interfering RNAs (siRNAs) targeting myostatin were cotransfected with a myostatin-expressing plasmid into HEK293 cells and identified for myostatin silencing by Western blot. Corresponding shRNAs were cloned into plasmid shRNA expression vectors. Myostatin or a randomer negative control shRNA plasmid was injected and electroporated into the tibialis anterior or its contralateral muscle, respectively, of nine rats that were sacrificed after 2 weeks. Six other rats received a beta-galactosidase reporter plasmid and were sacrificed at 1, 2, and 4 weeks. Uptake of plasmid was examined by beta-galactosidase expression, whereas myostatin expression was determined by real-time polymerase chain reaction (PCR) and Western blotting. Muscle fiber size was determined by histochemistry. Satellite cell proliferation was determined by PAX7 immunohistochemistry. Myosin heavy chain type II (MHCII) expression was determined by Western blot.Results P-Galactosidase reporter plasmid was expressed at I and 2 weeks but diminished by 4 weeks in tibialis anterior skeletal muscle. Myostatin shRNA reduced myostatin mRNA and protein expression by 27 and 48%, respectively. Tibialis anterior weight, fiber size, and MHCII increased by 10, 34, and 38%, respectively. Satellite cell number was increased by over 2-fold.Conclusions This is the first demonstration that myostatin shRNA gene transfer is a potential strategy to increase muscle mass. Copyright (c) 2006 John Wiley & Sons, Ltd.