乌鸡肌卫星细胞的分离培养与鉴定

Isolation, Culture and Identification of Muscle Satellite Cells of Black-bone Chicken

通过建立一种有效的乌鸡肌卫星细胞分离、培养及鉴定体系,为体外探讨家禽骨骼肌生长发育调控机制及骨骼肌损伤后修复的研究奠定基础。选取10日胚龄的乌鸡鸡胚为材料,采用Ⅰ型胶原酶和胰酶联合消化,差速贴壁法分离鸡肌卫星细胞,通过形态学识别和检测肌卫星细胞标志基因的表达等方法鉴定分离获得的细胞,并在低浓度血清培养基中诱导细胞成肌分化。结果显示:分离获得的细胞形态与肌卫星细胞相似;实时荧光定量PCR(q PCR)和蛋白免疫印迹法(Western blot)检测卫星细胞特异基因Paired protein box 7(Pax7)呈高水平表达;经成肌诱导分化后,细胞融合形成肌管,细胞免疫荧光法检测成肌分化标志基因肌球蛋白重链(Myosin heavy chain,My HC)呈阳性表达。研究成功从鸡胚中分离获得肌卫星细胞,并具有良好的体外增殖和分化能力,为肌肉的发育和损伤修复研究提供良好的细胞模型。

This study was aimed to establish an effective system to isolate, identify and culture chicken muscle satellite ceils, and provide foundational materials for exploring skeletal muscle growth, development and injury repair mechanism in vitro. Ten-day-age Lueyang black-bone chicken embryos were digested by eollagenase and trypsinase and isolated via differential adhesion. Then, the isolated cells were detected by morphological recognition andthe marker genes expression, induced to form myotubes in low concentration of FBS medium. The morphological characteristic of the isolated cells was similar to that of the muscle satellite cells, and qPCR and Western blot results showed high expression level of satellite cell specific gene PaxT. After induced differentiation, formation of myotubes was occured via cells fusion. In addition, the expression of MyHC as myogenic differentiation marker gene was detectable by cell immunofluorescence assay. This study successfully isolated and identified chicken muscle satellite cells with the ability to proliferate and differentiate in vitro. Thus this study provided an appropriate cell model for muscle development and injury repair research.