鲁西黄牛胚胎骨骼肌卫星细胞分离培养及生物学特性

Isolation and biological characteristics of skeletal muscle satellite cells of Luxi cattle embryo

为研究鲁西黄牛胚胎骨骼肌卫星细胞生物学特性,取30～50日龄鲁西黄牛胚胎四肢骨骼肌,分离培养鲁西黄牛胚胎骨骼肌卫星细胞。结果显示,试验成功地分离了鲁西黄牛胚胎骨骼肌卫星细胞,原代培养并传代至16代,免疫细胞化学染色检测卫星细胞表面特异性标志Desmin、MyoD呈阳性表达,RT-PCR检测Desmin、c-Met、Myf5、pax7呈阳性表达;绘制生长曲线,结果说明细胞具有较强增殖能力;应用流式细胞仪对细胞进行周期分析,结果显示S期细胞所占比例为8.94%;骨骼肌卫星细胞向成肌细胞诱导10d后,免疫细胞化学染色检测成肌特异性标志骨骼肌肌球蛋白呈阳性表达;骨骼肌卫星细胞向脂肪细胞诱导7d后,油红O染色呈阳性,RT-PCR检测脂肪细胞特异性标志pparγ、LPL呈阳性表达。结果表明试验成功建立了鲁西黄牛胚胎骨骼肌卫星细胞的体外扩增培养体系,且该骨骼肌卫星细胞有发育形成肌管和脂肪细胞的多项分化潜能。

To better understand the biological characteristics of Luxi cattle embryo skeletal muscle satellite cells,those cells from the skeletal muscles of the limbs of 30-50 days old Luxi cattle embryo were isolated and cultured.The results showed that two surface antigen markers,Desmin and Myod,were positively expressed in the harvested cells by immunofluorescence.Desmin,Myf5,c-Met and pax7 were found to be positively expressed by RT-PCR.Growth curve indicated that the cells were capable of proliferating strongly.In addition,the cell cycle analysis showed that the cells in S phase accounted for 8.94% of the total.After 10 days for myoblasts induce,the maker specific for fast skeletal myosin of myotubules was positively detected by immunofluorescence.After 7 days for adipogenic induce,lipid droplets were detected to be positive using Red Oil O dye;the adipocyte specific genes,ppar-γ and LPL,were identified to be positively expressed by RT-PCR.The Luxi cattle embryo skeletal muscle satellite cells were cultured successfully via vitro culture system,and could be induced to differentiate into multinucleated myotubes and adipocyte,respectively.This provided a new method for preserving animal genetic resources and for study stem cells theoretically in the future.