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目的分离培养鲁西黄牛胚胎骨骼肌卫星细胞,并研究其生物学特性。方法取30~50 d龄鲁西黄牛胚胎四肢骨骼肌,联合Ⅰ型胶原酶和胰蛋白酶及差速贴壁法分离获得骨骼肌卫星细胞,通过免疫细胞化学染色和RT-PCR检测卫星细胞表面标志desmin、MyoD、c-Met、Myf5、pax7,并研究其生物学特性。结果鲁西黄牛卫星细胞传代培养至15代以上,冻存后细胞活率为97.90%±0.96%;免疫细胞化学染色检测卫星细胞标记物desmin,MyoD呈阳性表达;RT-PCR检测desmin、c-Met、Myf5、pax7呈阳性表达;克隆形成率为56.39%±1.41%;染色体核型分析显示分离培养的细胞均来源于正常牛胚胎个体;通过不同的诱导剂将牛卫星细胞成功地诱导为成骨细胞和神经元样细胞,分别进行茜素红,甲苯胺蓝染色呈阳性表达,RT-PCR检测成骨细胞标记物osteopontin,Ⅰ型胶原蛋白和神经细胞标记物MAP2和nestin均呈阳性表达。结论本试验成功地分离获得了牛卫星细胞,并具有自我更新增殖和多向分化潜能。
Objective To isolate and culture Skeletal muscle satellite cells from Luxi cattle and to study its biological characteristics. Methods Skeletal muscle satellite cells were isolated from skeletal muscle of four limbs of Luxi cattle embryos at 30 ~ 50 d old age. Skeletal muscle satellite cells were isolated by collagenase Ⅰ, trypsin and differential adhesion method. Immunocytochemical staining and RT-PCR were used to detect satellite cell surface markers desmin, MyoD, c-Met, Myf5, pax7, and study their biological characteristics. Results Luxi cattle satellite cells were subcultured for more than 15 passages and the viability after cryopreservation was 97.90% ± 0.96%. Immunocytochemical staining showed that the satellite cell markers desmin and MyoD were positive. The expression of desmin and c- Met, Myf5 and pax7 were positive. The clonogenic rate was 56.39% ± 1.41%. The chromosome karyotype analysis showed that all the isolated cells were derived from normal bovine embryos. The bovine satellite cells were successfully induced by different inducing agents Osteoblasts and neuron-like cells were stained with alizarin red and toluidine blue respectively. Osteopontin, collagen Ⅰ and MAP2 and nestin were detected by RT-PCR. Conclusion The experiment successfully isolated and obtained cattle satellite cells, and has the self-renewal proliferation and multi-directional differentiation potential.