小鼠原代成肌细胞分离、纯化及培养

目的建立小鼠原代成肌细胞分离、纯化及培养方法 ,并诱导其成肌分化,检测成肌分化过程中微小RNA-1（miRNA-1）和miRNA-155表达情况。方法提取新生小鼠后腿肌肉,经胶原酶消化,利用细胞贴壁差异性分离及纯化原代成肌细胞。使用含马血清的分化培养基诱导其成肌分化,通过肌球蛋白重链抗体——MF20免疫荧光染色鉴定成肌能力。使用Taqman实时定量聚合酶链反应检测原代成肌细胞分化过程中miRNA-1和miRNA-155表达情况。结果成功分离并纯化小鼠原代成肌细胞;成功诱导原代成肌细胞向骨骼肌分化,MF20免疫荧光染色呈阳性。原代成肌细胞在成肌分化过程中miRNA-1表达明显上升,miRNA-1...

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Published in现代医药卫生 Vol. 33; no. 3; pp. 337 - 339
Main Author 熊雷 何衍佶 戴威 赵圆 高彦飞 邓忠良 聂茂
Format Journal Article
LanguageChinese
Published 重庆医科大学附属第二医院骨科,重庆,400016 2017
Subjects
微RNAs
成肌细胞
细胞,培养的
细胞分化
细胞,培养的
成肌细胞
细胞分化
MicroRNAs
Cells,cultured
Cell differentiation
微RNAs
Myoblasts
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Summary:目的建立小鼠原代成肌细胞分离、纯化及培养方法 ,并诱导其成肌分化,检测成肌分化过程中微小RNA-1（miRNA-1）和miRNA-155表达情况。方法提取新生小鼠后腿肌肉,经胶原酶消化,利用细胞贴壁差异性分离及纯化原代成肌细胞。使用含马血清的分化培养基诱导其成肌分化,通过肌球蛋白重链抗体——MF20免疫荧光染色鉴定成肌能力。使用Taqman实时定量聚合酶链反应检测原代成肌细胞分化过程中miRNA-1和miRNA-155表达情况。结果成功分离并纯化小鼠原代成肌细胞;成功诱导原代成肌细胞向骨骼肌分化,MF20免疫荧光染色呈阳性。原代成肌细胞在成肌分化过程中miRNA-1表达明显上升,miRNA-155表达明显下降,差异均有统计学意义（P〈0.05）。结论成功构建了提取小鼠原代成肌细胞的方法,为进一步研究成肌分化过程中miRNA基因调控提供了体外模型。
Bibliography:Xiong Lei,He Yanji,Dai Wei,Zhao Yuan,Gao Yanfei, Deng Zhongliang,Nie Mao A （ Department of Orthopedic Surgery, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China）
Objective To establish the methods of isolation,purification and culture of primary mouse myoblast,then to induce its myogenic differentiation and to detect the expression of microRNA-1（miRNA-1） and mi RNA-155 during this process.Methods The primary mouse myoblast was isolated and purified from the hind leg of neonatal mouse with collagenase digestion,and difference of cell adherence. Its myogenic differentiation was induced by the differentiation culture medium containing horse serum. The myogenic ability of primary myoblast was identified by myosin heavy chain antibody（MF20） immunofluorescence staining. The expression of mi RNA-1 and mi RNA-155 during the myogenic differentiation was detected by Taqman real time PCR.Result The primary mouse myoblast was successfully isolated and purified;the myogenic differentiation o
ISSN:1009-5519
DOI:10.3969/j.issn.1009-5519.2017.03.007