Key Laboratory of Mammalian Reproductive Biology and Biotechnology Ministry of Education, Inner Mongolia University, Inner Mongolia, Hohhot 010021, Peoples Republic of China.
Cell Biol Int. 2012 Jun 1;36(6):579-87. doi: 10.1042/CBI20110487.
Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.
骨骼肌卫星细胞是一种成年肌肉源性干细胞，受到越来越多的关注。绵羊卫星细胞在代谢、寿命、增殖和分化方面与人类卫星细胞更为相似，而不是大鼠和小鼠的卫星细胞。我们使用两步酶消化和差速贴壁法分离和纯化绵羊骨骼肌卫星细胞，对细胞进行鉴定并诱导分化，以检验其多能性。分离绵羊骨骼肌卫星细胞最有效的方法是Ⅰ型胶原酶和胰蛋白酶两步消化法，体外培养的最佳条件是含 20% FBS+10%马血清的培养基。免疫荧光染色显示卫星细胞表达 Desmin、α-横纹肌肌动蛋白、MyoD1、Myf5 和 PAX7。在成肌诱导后，多能性标志物 MyoG 和快肌肌球蛋白表达阳性，表明形成了多核肌管。在成骨诱导后，细胞表达骨钙素，茜素红和碱性磷酸酶（ALP）染色结果均为阳性。在成脂诱导后，细胞表达过氧化物酶体增殖物激活受体γ2（PPARγ2），细胞周围出现清晰的脂滴，油红 O 染色阳性。综上所述，成功建立了绵羊骨骼肌卫星细胞的分离、纯化和鉴定体系。
