巴什拜羊骨骼肌卫星细胞分离培养及诱导分化

为了获得巴什拜羊骨骼肌卫星细胞,本研究采集出生1日龄巴什拜羔羊后肢骨骼肌组织,采用两步酶消化法结合差速贴壁法分离纯化巴什拜羊骨骼肌卫星细胞,并对分离获得的骨骼肌卫星细胞进行了鉴定、传代培养及诱导分化等研究。结果表明,本研究采用的分离纯化方法可以高效获得巴什拜羊骨骼肌卫星细胞,RT-PCR检测结果表明骨骼肌卫星细胞标志性基因pax7、Myf5、MyoD、desmin和c-Met均呈阳性表达。获得的骨骼肌卫星细胞具有较强的增殖能力,连续传代12代,细胞的形态仍保持正常,且细胞的克隆形成率仍保持在50%以上,但是当细胞传代至第18代时,逐渐表现出较为明显的衰退。细胞的生长符合典型的"S"型生长曲线,且第2代和第8代细胞的生长曲线没有明显的差异,至第14代时细胞的增殖速度逐渐降低。采用低浓度马血清培养体系,可成功诱导巴什拜羊骨骼肌卫星细胞向肌管方向分化,诱导培养至第5天时,骨骼肌卫星细胞分化标志基因MyHC呈阳性表达。由此得出结论,本研究采用的骨骼肌卫星细胞分离纯化体系高效、可靠,可以满足较高纯度巴什拜羊骨骼肌卫星细胞的分离培养。

Isolated Culture and Induced Differentiation of Bashbay Sheep(Ovis aries) Skeletal Muscle Satellite Cells

To obtain skeletal muscle satellite cells of Bashbay sheep, hindlimb skeletal muscle of one day old Bashbay sheep were collected, two steps enzyme digestion method and differential adhesion method were used to isolate and purify skeletal muscle satellite cells of Bashbay sheep, and the identification, subculture, induced differentiation were investigated.The result showed that the separation and purification methods adopted in this study could obtain skeletal muscle satellite cells of Bashbay sheep efficien tly. The marker genes, pax7, Myf5, Myo D, desmin and c-Met, all showed positive expression in skeletal muscle satellite cells by RT-PCR detection. The obtained skeletal muscle satellite cells had strong proliferation ability, when they were sub cultured for 12 generations, they still kept normal shape, and the colony formation efficiency still kept upon 50 percent. But when the cells were sub cultured for 18 generations, they showed obvious decay. The proliferation curve of cells were typical S-type curve, and there were no significant difference between the proliferation curve of 2nd and 8th generation cell, but when the cells were sub cultured more than 14 generations, their proliferation speed gradually decreased. Applying low concentration horse serum system, skeletal muscle satellite cells of Bashbay sheep could be induced into myotubes successfully, and when the cells were induced for 5 days, the marker gene for the differentiation of skeletal muscle satellite cells, My HC showed positive expression. A conclusion is drawn that the system adopted for separation and purification of skeletal muscle satellite cells could isolate and culture skeletal muscle satellite cells of Bashbay sheep with high purity efficiently and reliably.