秦川牛胎儿骨骼肌卫星细胞的分离培养

Isolation and transfection of skeletal muscle satellite cells from Qinchuan fetal bovine

【目的】探索胎牛骨骼肌卫星细胞分离、培养、鉴定及基因转染的方法。【方法】分别采用Ⅰ型胶原酶消化、Ⅰ型胶原酶与胰蛋白酶二步消化及链霉蛋白酶消化法对胎牛骨骼肌卫星细胞进行培养,比较了3种不同分离方法所得细胞数及其存活率的差异;利用差速贴壁法和Percoll密度梯度离心相结合的方法纯化骨骼肌卫星细胞,对纯化后的细胞进行myostatin基因RT-PCR以及结蛋白(Desmin)免疫细胞化学染色鉴定,最后通过电转染法对纯化的骨骼肌卫星细胞进行EGFP基因转染研究。【结果】3种消化培养方法中,以链霉蛋白酶消化法分离得到的胎牛骨骼肌卫星细胞数显著高于其它2种方法(P<0.05),但细胞存活率较低(P<0.05);而采用Ⅰ型胶原酶与胰蛋白酶二步消化法可以得到相对较高的细胞数及存活率。利用差速贴壁和Percoll密度梯度离心相结合的方法可以得到纯化的骨骼肌卫星细胞;电转染法适用于骨骼肌卫星细胞的基因转染。【结论】建立了胎牛骨骼肌卫星细胞分离、培养、纯化、鉴定及基因转染的方法,为通过转基因方法改良秦川牛产肉性能研究奠定了基础。

【Objective】 The study was done to establish a stable method for isolation,purification and gene transfection of Qinchuan fetal bovine skeletal muscle satellite cells.【Method】 Fetal cattle muscle satellite cells were isolated from Semimembranosus muscle by digesting with collagenase Ⅰ,collagenase Ⅰ combined with trypsin（two-step methods） or pronase.The differences in cells number and survival rate were compared.The cells were purified by Percoll density gradient centrifugation combined with different adherent methods.The cells were characterized by RT-PCR for myostatin gene and immunocytochemistry for Desmin.Then EGFP gene was introduced by electric transfection method.【Result】 The results showed that the cells number isolated by pronase was higher than that by other methods（P〈0.05）,but the survival rate of cells was lower（P〈0.05）.The cells number and survival rate were higher by two-step methods.Purified skeletal muscle satellite cells were obtained by Percoll density gradient centrifugation combined with different adherent methods.Furthermore,the cells were successfully transfected by electric transfection method.【Conclusion】 We established the methods for isolation,culture,purification,characterization and gene transfection for Qinchuan fetal bovine skeletal muscle satellite cells and laid the foundation for the study of improving Qinchuan bovine meat production performance by transgenic methods.