雷帕霉素抑制体外成肌细胞分化过程中的肌管肥大并维持 C2C12 细胞的活力。

Rapamycin represses myotube hypertrophy and preserves viability of C2C12 cells during myogenesis in vitro.

机构信息

1 Institute of Resource Biology and Biotechnology, Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China. 2 Key Laboratory of Molecular Biophysics Ministry of Education, Huazhong University of Science and Technology, Wuhan, China. 3 Wuhan Institute of Biotechnology, Wuhan, China. 4 Department of Chemical Engineering and Food Science, Hubei University of Arts and Science, Hubei, China. 5 Address correspondence to: Mingzhang Ao, Ph.D., Longjiang Yu, Ph.D., College of Life Science and Technology, Huazhong University of Science and Technology, No. 1037 Luoyu Road, Wuhan 430074, China.

出版信息

Transplantation. 2014 Jul 27;98(2):139-47. doi: 10.1097/TP.0000000000000175.

DOI:10.1097/TP.0000000000000175

PMID:24926828

Abstract

BACKGROUND

Rapamycin (RAPA) has been successfully used for myoblast allotransplantation in X chromosome-linked muscular dystrophy mice. However, the mechanism of skeletal myogenesis, particularly in starved condition by RAPA, remains elusive. For this reason, we investigated the effect of RAPA on C2C12 myogenesis in serum-starved condition.

METHODS

Serum-free treated C2C12 cells were mimicked as skeletal myogenesis in nutrition shortage microenvironment. A methylthiazoletetrazolium (MTT) assay was used to investigate different RAPA concentrations on serum-free treated C2C12 cells and the following assays were used to detect the characteristic of C2C12 myogenesis by RAPA in vitro.

RESULTS

We found that 150 ng/mL of RAPA did not significantly suppress the viability of C2C12 differentiated cells by MTT assay. The RAPA concentration could protect myoblast serum-starved cells effectively from apoptosis through flow cytometry and retain myogenic regulatory factors through quantitative polymerase chain reaction analysis. However, RAPA significantly suppressed cell migration in wound healing assay (P<0.05). Morphological analyses indicated that RAPA also significantly suppressed myotube hypertrophy in serum-starved C2C12 cells. Western blot analysis revealed that the ratio of phosphate extracellular signal-regulated kinase/extracellular signal-regulated kinase and the protein level of p-Akt decreased in the proliferation medium and in the differentiation medium, respectively.

CONCLUSION

These findings suggest that myoblast cells are sensitive to RAPA under a serum-starved microenvironment. As an immunosuppressive agent, RAPA shall be used as a considering dosage and as a safe strategy for future myoblast allotransplantation.

摘要

背景

雷帕霉素（RAPA）已成功用于 X 染色体连锁肌营养不良症小鼠的成肌细胞同种异体移植。然而，其成肌机制，特别是在 RAPA 饥饿条件下的成肌机制，仍难以捉摸。基于此，我们研究了 RAPA 对饥饿条件下 C2C12 成肌的影响。

方法

无血清处理的 C2C12 细胞被模拟为营养缺乏微环境中的骨骼肌成肌。噻唑蓝（MTT）测定法用于研究不同浓度 RAPA 对无血清处理的 C2C12 细胞的影响，以下实验用于检测 RAPA 对体外 C2C12 成肌的影响。

结果

我们发现，150ng/ml 的 RAPA 并未通过 MTT 测定显著抑制 C2C12 分化细胞的活力。RAPA 浓度可通过流式细胞术有效保护肌母细胞免受血清饥饿诱导的细胞凋亡，并通过定量聚合酶链反应分析保留成肌调节因子。然而，RAPA 显著抑制划痕愈合试验中的细胞迁移（P<0.05）。形态学分析表明，RAPA 还显著抑制了血清饥饿的 C2C12 细胞中的肌管肥大。Western blot 分析显示，增殖培养基中磷酸化细胞外信号调节激酶/细胞外信号调节激酶的比值和分化培养基中 p-Akt 的蛋白水平分别降低。