兔骨骼肌卫星细胞的分离、培养及成肌特性

Separation and culture of rabbit skeletal muscle satellite cells and evaluation of their myotube formation capability in vitro

目的 建立高纯度、高丰度的兔骨骼肌卫星细胞的分离、培养方法,并对培养的肌卫星细胞体外增殖和成肌特性进行观察.方法 采用改良Ⅰ型胶原酶和胰蛋白酶混合消化法结合差速贴壁培养以分离、纯化1周龄幼兔小腿三头肌来源的骨骼肌卫星细胞.镜下观察肌卫星细胞生长形态,结蛋白（Desmin）免疫组化染色鉴定体外培养的肌卫星细胞,MTT法绘制细胞生长曲线,并对分离培养的肌卫星细胞成肌特性进行诱导培养观察.结果 差速贴壁后的悬浮肌卫星细胞折光性强,呈透亮球形,数量较多,贴壁生长后逐渐变为长梭形;免疫组化染色检测分离培养的细胞高表达Desmin,证明为肌卫星细胞,纯度高达95％;传代培养的肌卫星细胞24h后开始增殖,第3～7天处于对数生长期,第8天后进入平台期;不经诱导分化培养基培养的肌卫星细胞也可融合形成肌管,但形成肌管速度不及诱导分化培养组快速.结论 成功建立了具有操作简便、不易污染和纯度高的兔骨骼肌卫星细胞分离、培养方法,为采用肌卫星细胞为种子细胞的再生医学研究奠定实验基础.

Objective To establish the methods for the separation and culture of rabbit skeletal muscle satellite cells and evaluate their myotube formation capability. Methods Skeletal muscle satellite cells from 1 - week - old rabbit triceps surae were isolated and pured by improved collagenase - I and trypsin digestion using different adhesion method. The morphology of satellite cells was observed under microscope. Desmin immunohistochemi- eal staining was used for the identification of satellite cells. MTY assay was used for drawing the cell growth curve. The induction culture and characteristics of myotube formationin vitro was observed. Results After twice differential adherence, the number of suspended cells with strong refraction was increased rapidly, and cells gradually became long- shuttle in shape. The strong positive expression was detected by desmin immunohisto- chemical staining. The purity was up to 95%. The satellite cells began to proliferate after 24 h. It was in loga- rithmic growth and platform phase after subeuhured for 3rd -7th day and 8th day, respectively. Without the in- duced differentiation culture medium, the satellite cells could still fuse to form myotubes. However, the speed of myotube formation was slower than that with induced differentiation culture medium. Conclusion This experiment has successfully established methods for separation and culture of rabbit skeletal muscle satellite ceils with easy operation, little pollution and high purity, which provide the basis for the experimental foundation of regeneration medicine research.