| 摘要: |
| 为建立肉鸡成肌细胞的体外分离、培养及鉴定体系，选取11 d鸡胚为材料，利用Ⅰ型胶原酶消化鸡胚胸肌，差速贴壁法分离纯化成肌细胞，优化成肌细胞培养条件；通过细胞形态学观察和成肌分化标志基因表达情况进行细胞鉴定，并利用含2%马血清的培养基诱导其成肌分化。结果表明:分离获得的细胞贴壁后呈纺锤形，39.5 ℃培养条件下细胞增殖速度显著高于37.0 ℃(P<0.05)；经实时荧光定量PCR(RT-qPCR)检测分析，增殖期与分化期均有成肌细胞的特异性标志基因成肌因子5(Myogenic factor 5，Myf5)与成肌分化因子1(Myogenic differentiation 1，MyoD)的表达，且与增殖期相比，成肌细胞分化期肌细胞生成素(Myogenin，MyoG)与肌肉调节因子4(Muscle regulatory factor，MRF4)基因表达量显著升高(P<0.05)；此外，细胞增殖期结蛋白(Desmin)和分化期肌球蛋白重链(Myosin heavy chain，MyHC)呈阳性表达。综上，成功分离获得了肉鸡成肌细胞，并在39.5 ℃培养条件下具有更好的增殖能力，为研究肌肉发育及其营养调控机制奠定了基础。 |
| 关键词: 肉鸡 成肌细胞 鉴定 培养温度 成肌诱导分化 |
| DOI：10.11841/j.issn.1007-4333.2020.09.08 |
| 投稿时间：2020-01-06 |
| 基金项目:国家自然科学基金项目(31702129)；安徽省自然科学基金项目(1808085QC62)；安徽省科技重大专项(17030701006) |
|
| Isolation, culture and identification of primary myoblasts from broiler |
|
CHEN Kaikai,ZHANG Cheng,ZHAO Fei,WANG Chi,WANG Xiaocheng,GENG Zhaoyu,JIANG Runshen
|
| (College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China) |
| Abstract: |
| To establish a detection system for isolation, culture and identification of myoblasts from broiler in vitro, eleven-day-old broiler embryos were digested with type I collagenase and isolated via differential adhesio, followed by the optimization of cultivation conditions. The isolated cells were then identified by cell morphology observation and myogenic differentiation marker gene expression and induced to myogenic differentiation in a 2% horse serum. The results showed that the myoblasts were spindle-shaped, and the cell proliferation rate was significantly higher at 39. 5 ℃ than that at 37. 0 ℃(P<0. 05). Real-time quantitative PCR analysis showed that the myoblast-specific marker genes myogenic differentiation marker 1(MyoD)and myogenic factor 5(Myf5)were expressed at both the proliferation and differentiation stages. And the expression of myogenin(MyoG)and muscle regulatory factor(MRF4)in the differentiated myoblasts was significantly higher than that in the proliferation phase(P<0. 05). In addition, the cells were positive for desmin at proliferation stage and myosin heavy chain(MyHC)antibody at differentiation stage by immunofluorescence assay. In conclusion, the broiler primary myoblasts were successfully isolated and had better proliferation ability at 39. 5 ℃. This study provided cellular foundation for exploring the mechanism of muscle development and its nutritional regulation. |
| Key words: broiler myoblast identification culture temperature myogenic differentiation |
