抑制肌肉生长抑制素可促进大鼠骨髓间充质基质细胞的成肌分化。

Inhibition of myostatin promotes myogenic differentiation of rat bone marrow-derived mesenchymal stromal cells.

机构信息

Neurology Department, The First Affiliated Hospital, Sun Yat-Sen University, Guang Zhou, PR China.

出版信息

Cytotherapy. 2009;11(7):849-63. doi: 10.3109/14653240903131632.

DOI:10.3109/14653240903131632

PMID:19903098

Abstract

BACKGROUND AIMS

Mesenchymal stromal cells (MSC) have been thought to be attractive candidates for the treatment of Duchenne muscular dystrophy (DMD), but the rate of MSC myogenesis is very low. Thus MSC treatment for DMD is restricted. Myostatin (Mstn), a negative regulator of myogenesis, is known to be responsible for limiting skeletal muscle regeneration. We hypothesized that inhibition of Mstn by using anti-Mstn antibody (Ab) would ameliorate the myogenic differentiation of MSC in vitro and in vivo.

METHODS

MSC were isolated from rat bone marrow. Induced rat MSC (rMSC) were treated with various concentrations of anti-Mstn Ab. The expression of myogenic differentiation antigen (MyoD), myogenin and myosin heavy chain-type alpha (MHC-alpha) were estimated by immunofluorescence analysis and reverse transcription-polymerase chain reaction (RT-PCR). Adipogenic differentiation of rMSC inhibited by anti-Mstn Ab was evaluated by Oil Red O staining. The expression of dystrophin was detected 16 weeks after anti-Mstn Ab injection and rMSC transplantation by immunofluorescence staining, RT-PCR and Western blot. Motor function, serum creatine kinase (CK) and histologic changes were also evaluated.

RESULTS

Five-azacytidine-mediated myogenic differentiation induced significant endogenous Mstn expression. Anti-Mstn Ab improved the expression of MyoD, myogenin and MHC-alpha and inhibited adipocyte formation. Sixteen weeks after transplantation, the inhibition of Mstn had improved motor function and muscle mass. In accordance with the increased motor function and muscle mass, dystrophin expression had increased. Furthermore, serum CK and centrally nucleated fiber (CNF) levels decreased slightly, suggesting specific pathologic features of the dystrophic muscle were partially restored.

CONCLUSIONS

Using anti-Mstn Ab, we found that inhibition of Mstn improved myogenic differentiation of rMSC in vitro and in vivo. A combination of Mstn blockade and MSC transplantation may provide a pharmacologic and cell-based strategy for the treatment of DMD.

摘要

背景目的

间充质基质细胞（MSC）被认为是治疗杜氏肌营养不良症（DMD）的有吸引力的候选者，但 MSC 的成肌作用率非常低。因此，DMD 的 MSC 治疗受到限制。肌肉生长抑制素（Mstn）是成肌的负调节剂，已知其负责限制骨骼肌再生。我们假设使用抗 Mstn 抗体（Ab）抑制 Mstn 会改善 MSC 在体外和体内的成肌分化。

方法

从大鼠骨髓中分离 MSC。诱导大鼠 MSC（rMSC）用不同浓度的抗 Mstn Ab 处理。通过免疫荧光分析和逆转录-聚合酶链反应（RT-PCR）估计肌生成分化抗原（MyoD）、肌生成素和肌球蛋白重链型 alpha（MHC-alpha）的表达。通过油红 O 染色评估 rMSC 成脂分化受抗 Mstn Ab 抑制的情况。通过免疫荧光染色、RT-PCR 和 Western blot 检测 16 周后抗 Mstn Ab 注射和 rMSC 移植后抗肌萎缩蛋白的表达。还评估了运动功能、血清肌酸激酶（CK）和组织学变化。

结果

5-氮杂胞苷介导的成肌分化诱导了显著的内源性 Mstn 表达。抗 Mstn Ab 改善了 MyoD、肌生成素和 MHC-alpha 的表达，并抑制了脂肪细胞形成。移植 16 周后，Mstn 的抑制改善了运动功能和肌肉质量。与运动功能和肌肉质量的增加相吻合，肌萎缩蛋白的表达增加了。此外，血清 CK 和中央核纤维（CNF）水平略有下降，表明营养不良肌肉的特定病理特征得到了部分恢复。