成年大鼠骨骼肌干细胞的制备与新型培养方法

 目的：建立高效分离和扩增成年大鼠骨骼肌干细胞的实验方法。方法：通过混合酶消化法从少量成年大鼠骨骼肌样品中分离出骨骼肌干细胞；采用悬浮培养法培养获得骨骼肌干细胞团,批量扩增骨骼肌干细胞；用qRT-PCR和免疫细胞化学染色检测干细胞标志物Nanog、Oct4和骨骼肌干细胞标志物肌源性因子5（myogenic factor 5，Myf5）、配对盒蛋白7 (paired box protein 7,Pax7)的表达；成脂或成骨分化诱导培养基诱导检测骨骼肌干细胞的多功能性。结果：采用混合酶消化法可获得骨骼肌干细胞单细胞，在悬浮的培养条件下能快速克隆成骨骼肌细胞团,贴壁培养后能看到骨骼肌干细胞从细胞团中爬出来，和传统的差速贴壁方法相比悬浮培养法获得的骨骼肌干细胞纯度更高，细胞状态更好，干性因子Nanog、Oct 4和骨骼肌干细胞标志物Myf5、Pax 7的表达更高（P<0.05）；在不同培养基的诱导下能向骨骼肌、成骨和脂肪细胞系分化。结论：悬浮培养法能获得大批量干性好、纯度高、具有多向分化潜能的骨骼肌干细胞，从而为肌肉相关疾病的细胞治疗提供优良的种子细胞。

A new method for preparation of skeletal muscle stem cells in adult rats

AIM：To establish a method for effective isolation and amplification of skeletal muscle stem cells from adult rats. METHODS：Muscle stem cells were isolated from adult rat skeletal muscle by digesting with mixed enzymes. The cells were expanded into myosphere in suspension culture. The phenotypic characteristics and functional properties of these cells were determined by qRT-PCR and immunocytochemistry. The cells were cultured with adipogenic induction medium and osteogenic induction medium to identity their capacity of multi-lineage differentiation. RESULTS：A single muscle stem cell was obtained by digesting with mixed enzymes. The cell was expanded into myosphere in suspension culture, which highly expressed Nanog, Oct4, Myf5 and Pax7 compared with the traditional method. The muscle stem cells differentiated into skeletal myocytes, osteoblasts and adipocytes after cultured with induction media. CONCLUSION：In suspension culture, we can acquire highly purified muscle stem cells in large differentiation number with multi-lineage differentiation capacity.