大围山微型鸡骨骼肌卫星细胞的分离培养、鉴定及生物学特性研究

Isolation,Culture,Identification and Biological Characteristics of Skeletal Muscle Satellite Cells in Daweishan Mini Chicken in Yunnan Province

为研究大围山微型鸡骨骼肌卫星细胞的体外纯化、培养、鉴定方法及其生物学特性,试验采用Ⅰ型胶原酶和胰蛋白酶两步酶消化法结合差速贴壁技术分离纯化出云南大围山微型鸡骨骼肌卫星细胞,进行体外原代和传代培养。观察细胞各个阶段的形态,通过生长曲线、诱导分化等手段研究骨骼肌卫星细胞的增殖与分化能力,利用RT-PCR以及免疫细胞化学染色对所获得的细胞进行鉴定。结果显示:两步酶消化法适用于微型鸡骨骼肌卫星细胞的分离和获取,此法分离得到的微型鸡骨骼肌卫星细胞表达卫星细胞特异的标志基因Pax7。在生长培养基作用下,细胞增殖旺盛,经诱导分化后,细胞可融合成肌管。该试验方法对研究家禽骨骼肌细胞增殖、分化和骨骼肌生长、发育的细胞生物学机制具有一定科学意义。

In order to study the methods for purification,culture,identification of skeletal muscle satellite cells in Daweishan Mini chickens,and its biological characteristics,skeletal muscles from Daweishan Mini chickens were digested in collagenase type Ⅰ and individual myofibres dissociated by trituration and washed. The isolated myofibres were cultured primarily in medium to obtain the satellite cell- derived myoblasts. Morphological observation and growth curve were used to evaluate the proliferation and differentiation ability of skeletal muscle satellite cells of Mini chicken. The cells were further identified by RT-PCR and cellular immunochemical stain. The results showed that culture skeletal muscle satellite cells using collegenase-Ⅰ and trypsin was reliable protocol for collecting of Mini chicken′s skeletal muscle satellite cells due to the high expression of Pax7 gene in the satellite cells and with high proliferative and differentiation ability. The present study of skeletal muscle satellite cells culture provided an effective protocol to study the mechanism of myogenic proliferation,differentiation and muscle regeneration in avian stem cell physiological research.