反义RNA干扰MSTN对牛肌肉卫星细胞脂肪生成相关基因的影响

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摘要肌肉生长抑制素(myostatin, MSTN)属于转化生长因子β (transforming growth factor β, TGF-β)超家族成员，在骨骼肌中高表达；敲除MSTN后，肌肉质量增加、脂肪含量变少，但抑制MSTN表达后，如何影响脂肪沉积，尚未研究清楚。为了探究抑制MSTN表达后对脂肪相关基因表达的影响，本研究利用反义RNA技术，成功构建了双向表达载体pMSTN-CMV-CAG-antiMSTN，并通过Western blot筛选出抑制效果最佳的MSTN的反义载体。利用最佳反义MSTN载体在牛(Bos taurus)肌肉卫星细胞中有效干扰MSTN表达后，检测了脂肪合成相关基因(脂肪酸合酶(fatty acid synthase, FASN), 乙酰CoA羧化酶(acetyl-CoA carboxylase, ACC)和硬脂酰辅酶A去饱和酶1(stearoyl-CoA desaturase-1, SCD-1))、脂肪酸氧化分解相关基因(肉毒碱棕榈酰转移酶IB(carnitine palmitoyltransferase 1b, CPT-IB), 乙酰辅酶A氧化酶(acyl-CoA oxidase, ACOX1)和烯酰辅酶A水合酶(enoyl-CoA hydratase 1, ECHDC1))以及脂肪转运蛋白(fatty acid transport proteins, FATP)表达的变化。结果显示，在MSTN基因被干扰后，肌肉卫星细胞中脂肪酸合成相关基因表达量上调，脂肪氧化分解相关基因中ACOX1和CPT-1B表达量上调，而ECHDC1基因表达量下调，另外，FATP表达基本没有变化。MSTN功能缺失后，脂肪合成和脂肪氧化分解过程都得到了加强，所以MSTN敲除所导致的脂肪减少并不是由于抑制了脂肪合成，而是由于脂肪分解加强，为肌肉提供更多的能量。MSTN下调后，CPT-1B的表达上调最为显著，而CPT-1B是脂肪酸β氧化的关键基因，所以研究结果提示MSTN对于脂肪的影响主要是通过影响β氧化过程实现的。研究结果为MSTN下调所导致的脂肪含量下降提供了理论依据，为MSTN作用于脂肪沉积的机制做出了初步探讨。

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	李光鹏

关键词 ：肌肉生长抑制素(MSTN), 反义RNA, 牛肌肉卫星细胞, 脂肪沉积

Abstract：Myostatin (MSTN) is a member of the transforming growth factor β (TGF-β) superfamily, which plays a key role in muscle development. Numerous studies have shown that knockout of MSTN results in a widespread increase in skeletal muscle mass, and a significant reduction in fat accumulation. However, the mechanisms involved in fat deposition process yielded by MSTN gene suppression has not been understood. In the present study, we constructed 5 two-way expression antisense RNA vectors pMSTN-CMV-CAG-antiMSTN, then transfected bovine fetal fibroblast cells, and screened the best antisense RNA vector which significantly disturbed the MSTN expression in protein level with Western blot. The vectors were then confected to a bovine muscle satellite cells and the effect of MSTN knockdown to genes associated with adipogenesis such as fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase-1 (SCD-1), genes associated with lipolysis such as carnitine palmitoyltransferase 1b (CPT-1B), acyl-CoA oxidase(ACOX1) and enoyl-CoA hydratase 1 (ECHDC1), and fat transport protein (FATP) gene were investigated. The results showed that genes associated with adipogenesis and lipolysis were all up-regulated except the expression of ECHDC1 when MSTN knockdown in muscle satellite cells. The expression of gene associated with fat transport has no significant change. The results suggested that MSTN knockdown could enhance both adipogenesis and lipolysis, so the reduced fat mass resulted from MSTN knockdown was due to the increased lipolysis rather than inhibition of adipogenesis. And the expression of CPT-1B was up-regulated in the highest level among the genes investigated, this result suggested that the effect of MSTN to fat was mainly by the process of β oxidation, because CPT-1B was a main factor of β oxidation. This study provides a theoretical basis for the decreased fat content resulted from MSTN reduction, and makes a preliminary study for the effect of MSTN on fat deposition.

Key words： Myostatin (MSTN) Antisense RNA Bovine muscle satellite cells Fat deposition

收稿日期: 2016-06-20 出版日期: 2016-11-07

基金资助:国家科技重大专项“高产优质转基因肉牛新品种培育（2015ZX08007-002)”

通讯作者: 李光鹏 E-mail: gpengli@imu.edu.cn;gpengli@immu.edu.cn;imudwzx@163.com

引用本文:

高丽胡思敏谷明娟王丽荣扈廷茂李光鹏. 反义RNA干扰MSTN对牛肌肉卫星细胞脂肪生成相关基因的影响[J]. , 2016, 24(12): 1846-1854.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I12/1846

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