牛胚胎骨骼肌卫星细胞的分离、培养及诱导分化

Isolation,culture and differentiation of skeletal muscle satellite cells of Luxi cattle embryo

目的分离培养鲁西黄牛胚胎骨骼肌卫星细胞，并研究其生物学特性。方法取30～50d龄鲁西黄牛胚胎四肢骨骼肌，联合I型胶原酶和胰蛋白酶及差速贴壁法分离获得骨骼肌卫星细胞，通过免疫细胞化学染色和RT-PCR检测卫星细胞表面标志desmin、MyoD、c．Met、My￡5、pax7，并研究其生物学特性。结果鲁西黄牛卫星细胞传代培养至15代以上，冻存后细胞活率为97．90％±0．96％；免疫细胞化学染色检测卫星细胞标记物desmin，MyoD呈阳性表达；RT—PCR检测desmin、c—Met、My￡5、pax7呈阳性表达；克隆形成率为56．39％±1．41％；染色体核型分析显示分离培养的细胞均来源于正常牛胚胎个体；通过不同的诱导剂将牛卫星细胞成功地诱导为成骨细胞和神经元样细胞，分别进行茜素红，甲苯胺蓝染色呈阳性表达，RT-PCR检测成骨细胞标记物osteopontin，I型胶原蛋白和神经细胞标记物MAP2和nestin均呈阳性表达。结论本试验成功地分离获得了牛卫星细胞，并具有自我更新增殖和多向分化潜能。

Objective To isolate and culture skeletal muscle satellite cells of Luxi cattle embryo and study its biological characteristics. Methods The skeletal muscles were taken from the limbs of Luxi cattle embryo of 30 - 50 d old to isolate and culture the skeletal muscle satellite cells by differential adherence method and co-digestion of type I collagenase and trypsin. Satellite cell surface markers, desmin, MyoD, c-Met, Myf5 and pax？ were detected by immunocytochemistry and RT-PCR, and its biological characteristics were researched. Results The satellite cells were cultured to over passage 15. Cell viability was （97.90 ＋ 0.96） % after cryopreservation. The immunocytocbemical staining showed that the specific surface antigen markers desmin and MyoD were positive, and RT-PCR also indicated the positive expressions of desmin, MyfS, c-Met and pax？. Colony formation was （56.39 ＋ 1.41 ）%. The karyotype analysis demonstrated that the isolated satellite cells were all derived from normal cattle embryos. Bovine satellite cells were successfully induced into osteoblasts and neuron-like cells by various inductors, and were positively expressed after alizarin red and toluidine blue staining. The osteoblast specific genes osteopontin and type I collagen and the nerve cell specific genes MAP-2 and nestin were positive by RT-PCR. Conclusion The skeletal muscle satellite cells of the Luxi cattle embryo are successfully isolated and cultured in vitro , They have the ability of being differentiated into osteoblasts and neuron-like cells.