上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 技术与方法 • 上一篇    下一篇

骨骼肌C2C12细胞培养分化及肌纤维类型鉴定

周丽娜, 王 彦, 李庆云, 许华俊, 蓝孝斐, 张秀娟   

  1. 上海交通大学 医学院附属瑞金医院呼吸科， 上海 200025
  • 出版日期:2013-10-28 发布日期:2013-10-31
  • 通讯作者: 李庆云, 电子信箱: liqingyun68@hotmail.com。
  • 作者简介:周丽娜（1989—）， 女， 硕士生; 电子信箱: zhoulina1395@hotmail.com。
  • 基金资助:

    国家自然科学基金（81270148）

Culture and differentiation of C2C12 cells for identification of skeletal muscular fibers

ZHOU Li-na, WANG Yan, LI Qing-yun, XU Hua-jun, LAN Xiao-fei, ZHANG Xiu-juan   

  1. Department of Respiratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2013-10-28 Published:2013-10-31
  • Supported by:

    National Natural Science Foundation of China，81270148

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摘要：

目的 建立体外骨骼肌细胞培养分化与肌纤维鉴定技术模型。方法 取骨骼肌C2C12细胞，用含20 mL/L马血清的高糖DMEM培养基调节细胞浓度为5×104/mL，在孵育箱内分化培养成肌管细胞。采用免疫荧光技术检测肌细胞生成素（myogenin）的表达。在C2C12细胞成功分化为肌管细胞后分为内脏脂肪素（visfatin）刺激组（给予25 ng/mL visfatin刺激48 h）和对照组（不以visfatin刺激），分别采用RT-PCR与Western blotting技术检测肌球蛋白重链（MHC）亚型（MHC-Ⅰ、MHC-ⅡA和MHC-ⅡB）的相对表达量，并计算Ⅰ型纤维/Ⅱ型纤维的比例。结果 骨骼肌C2C12细胞分化前呈梭形，单核伴多个核仁，分化时逐渐变长，形成肌管，细胞核为多核，呈串珠样排列。免疫荧光显示myogenin表达于细胞核中，验证了C2C12细胞的分化潜能。RT-PCR和Western blotting检测均显示所培养的骨骼肌C2C12细胞在马血清诱导分化成肌管细胞后，具有表达3种MHC亚型mRNA和蛋白的能力，visfatin刺激组3种MHC亚型的mRNA和蛋白的表达水平均显著高于对照组，差异有统计学意义（P<0.05）。对照组和visfatin刺激组的Ⅰ型纤维/Ⅱ型纤维比例分别为0.63和0.60。结论 骨骼肌C2C12细胞模型可用于肌纤维分化及类型鉴定，并能对低浓度visfatin刺激产生有效反应，为后续visfatin引起上气道肌肉塌陷的机制研究奠定了基础。

关键词: C2C12细胞, 分化, 肌细胞生成素, 肌球蛋白重链, 肌纤维类型鉴定

Abstract:

Objective To establish a model for in vitro culture and differentiation of skeletal muscular cells and identification of muscular fibers. Methods C2C12 cells of skeletal muscle were obtained, 5×104/mL C2C12 cells were cultured in DMEM with 20 mL/L horse serum in a incubator for in vitro differentiation. The expression of myogenin was detected by immunofluorescence technique. The differentiated C2C12 cells were treated with 25 ng/mL visfatin for 48 h (visfatin treatment group) or not treated with visfatin (control group), the expression of mRNA and protein of myosin heavy chain (MHC) subtypes (MHC-Ⅰ, MHC-ⅡA and MHC-ⅡB) was detected by RT-PCR and Western blotting, and the ratios of expression of MHC-Ⅰ to MHC-Ⅱ were calculated. Results The undifferentiated spindle-like cells had only one nucleus with several nucleoli, while the differentiated cells became elongated with numerous nuclei clustering like a string. Immunofluorescence detection demonstrated that myogenin was expressed in the nucleus which confirmed the differentiation of C2C12 cells. RT-PCR and Western blotting indicated the capability of C2C12 cells to express mRNA and protein of three MHC subtypes after in vitro induced differentiation, and the expression of mRNA and protein of three MHC subtypes in visfatin treatment group was significantly higher than that in control group (P<0.05). The ratios of expression of MHC-Ⅰ to that of MHC-Ⅱ in control group and visfatin treatment group were 0.63 and 0.60 respectively. Conclusion C2C12 cell model can be used to induce the differentiation of skeletal muscle and identify the types of muscular fibers, and the cell model responses to visfatin at its lower concentration, which provides basis for the further research on the mechanism of upper airway collapse.

Key words: C2C12 cells, differentiation, myogenin, myosin heavy chain, identification of skeletal muscular fibers