鸽骨骼肌卫星细胞的分离、培养及成肌特性
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摘要
【目的】探讨白羽王鸽Columba livia骨骼肌卫星细胞的分离、培养和鉴定的方法,建立完整的家鸽骨骼肌卫星细胞的培养体系。【方法】选择孵化16 d的鸽胚作为试验材料,采用组织块贴壁法和胶原酶消化法分离胸肌的骨骼肌卫星细胞并绘制其生长曲线。待卫星细胞分化出肌管后,采用免疫荧光法检测肌球蛋白重链(MyHC)的表达;在细胞分化出肌管前、后分别提取总RNA,采用RT-qPCR方法检测Desmin、Pax7、MyoG和MyoD1基因在细胞分化出肌管前、后的相对表达量。【结果】组织块贴壁法和胶原酶法均能成功地分离出骨骼肌卫星细胞,其生长曲线呈"S"型;该细胞经含体积分数为20%FBS的DMEM高糖培养液培养7 d后视野中出现大量明显可见的肌管,成肌特异性标志MyHC表达呈阳性。RT-qPCR结果表明,Desmin和MyoG基因分化后的相对表达量分别是分化前的5.68和10.38倍,而Pax7和MyoD1基因分化前的相对表达量分别是分化后的7.01和5.51倍。【结论】建立了鸽骨骼肌卫星细胞的培养体系,为今后进行家鸽肌肉发育的研究提供细胞模型。
【Objective】 To investigate the method of isolation, culture and identification of white king pigeon(Columba livia) skeletal muscle satellite cells, and establish a complete culture system of pigeon skeletal muscle satellite cells. 【Method】 We selected 16-day pigeon embryos as experimental materials. Skeletal muscle satellite cells were isolated from the pectoral muscle by the tissue explants adherent method and collagenase digestion method, and the growth curve was drawn. MyHC expression was detected by immunofluorescence assay after differentiation of the satellite cells into myotubes. We detected mRNA expression of Desmin, Pax7, MyoG and MyoD1 genes before and after cell differentiation into myotubes using RT-qPCR. 【Result】 Skeletal muscle satellite cells were successfully isolated by the tissue explants adherent method and collagenase method, and cell growth appeared in S-shaped curve. After seven days of culture in high glucose DMEM containing 20% fetal bovine serum(FBS), a large number of visible myotubes were observed,and the myogenic differentiation marker MyHC was expressed in differentiated cells. RT-qPCR results showed that the relative expressions of Desmin and MyoG genes in differentiated myotube cells were 5.68 and 10.38 times of those in skeletal muscle satellite cells before differentiation, while the relative expressions of Pax7 and MyoD1 genes in skeletal muscle satellite cells before differentiation were 7.01 and 5.51 times of those in differentiated myotube cells, respectively. 【Conclusion】 The culture method of pigeon skeletal muscle satellite cells has been established,which provides a cell model for future studies of muscle development in pigeons.
【Objective】 To investigate the method of isolation, culture and identification of white king pigeon(Columba livia) skeletal muscle satellite cells, and establish a complete culture system of pigeon skeletal muscle satellite cells. 【Method】 We selected 16-day pigeon embryos as experimental materials. Skeletal muscle satellite cells were isolated from the pectoral muscle by the tissue explants adherent method and collagenase digestion method, and the growth curve was drawn. MyHC expression was detected by immunofluorescence assay after differentiation of the satellite cells into myotubes. We detected mRNA expression of Desmin, Pax7, MyoG and MyoD1 genes before and after cell differentiation into myotubes using RT-qPCR. 【Result】 Skeletal muscle satellite cells were successfully isolated by the tissue explants adherent method and collagenase method, and cell growth appeared in S-shaped curve. After seven days of culture in high glucose DMEM containing 20% fetal bovine serum(FBS), a large number of visible myotubes were observed,and the myogenic differentiation marker MyHC was expressed in differentiated cells. RT-qPCR results showed that the relative expressions of Desmin and MyoG genes in differentiated myotube cells were 5.68 and 10.38 times of those in skeletal muscle satellite cells before differentiation, while the relative expressions of Pax7 and MyoD1 genes in skeletal muscle satellite cells before differentiation were 7.01 and 5.51 times of those in differentiated myotube cells, respectively. 【Conclusion】 The culture method of pigeon skeletal muscle satellite cells has been established,which provides a cell model for future studies of muscle development in pigeons.
引文
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[12]李方华,侯玲玲,马月辉,等.北京油鸡骨骼肌卫星细胞的分离、培养、鉴定及成肌诱导分化的研究[J].中国农业科学,2010,43(22):4725-4731.
[13]单艳菊,束婧婷,宋迟,等.鸭骨骼肌卫星细胞的分离培养与鉴定[J].江苏农业科学,2012,40(12):26-28.
[14]樊廷俊,徐晓辉,姜国建,等.条斑星鲽连续性鳍细胞系的建立与鉴定[J].山东大学学报(理学版),2010,45(5):22-27.
[15]周海波,蔡建庭,李茂岚,等.白藜芦醇诱导人胃癌原代细胞裸鼠移植瘤凋亡的研究[J].中国病理生理杂志,2005,21(3):528-532.
[16]庄鹏,江元森,马会慧,等.长期培养的大鼠原代肝细胞功能和形态学观察[J].中国病理生理杂志,2005,21(5):1001-1005.
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[18]陈岩,王琨,朱大海.鸡骨骼肌卫星细胞的分离培养、鉴定及生物学特性研究[J].遗传,2006,28(3):257-260.
[19]夏家红,谢艾妮,徐磊,等.大鼠骨骼肌卫星细胞体外培养的实验研究[J].中华实验外科杂志,2005,22(2):214-215.
[20]TAKAYA T,NIHASHI Y,KOJIMA S,et al.Autonomous xenogenic cell fusion of murine and chick skeletal muscle myoblasts[J].Anim Sci J,2017,88(11):1880-1885.
[21]LAZARIDES E,HUBBARD B D.Immunological characterization of the subunit of the 100 A filaments from muscle cells[J].Proc Natl Acad Sci USA,1976,73(12):4344-4348.
[22]陈永乐,周光前,邓宇斌,等.C2C12成肌细胞体外诱导分化为肌管的实验[J].中山大学学报(医学科学版),2008,29(1):10-15.
[23]王静,宋新磊,崔焕先,等.骨骼肌卫星细胞的特性及其增殖与分化的调控[J].中国细胞生物学学报,2007,29(4):497-502.
[24]宋佳,曾缨,郑民缨,等.肌肉特异性微小RNA和成肌调节因子在C2C12细胞成肌分化过程中的表达[J].中国组织工程研究,2012,16(32):5999-6005.
[25]冯永强,李峥,褚万立,等.大鼠骨骼肌卫星细胞的原代培养鉴定和体外分化特点[J].中华医学杂志,2016,96(12):971-974.
[26]邱华玲,于建兴,陈宏权.MyoG基因结构与功能研究进展[J].生物信息学,2007,5(4):190-192.
[27]LING Y H,SUI M H,ZHENG Q,et al.miR-27b regulates myogenic proliferation and differentiation by targeting Pax3 in goat[J].Sci Rep,2018,8:3909.doi:10.1038/s41598-018-22262-4.
[2]卜柱.我国肉鸽产业发展现状及未来行情走势[J].中国禽业导刊,2015(22):24-27.
[3]ANDERSON J L,TAYLOR RL JR,SMITH E C,et al.Differentially expressed genes in aortic smooth muscle cells from atherosclerosis-susceptible and atherosclerosis-resistant pigeons[J].Poult Sci,2012,91(6):1315-1325.
[4]CUI J,HALBROOK R S,ZANG S,et al.Use of homing pigeons as biomonitors of atmospheric metal concentrations in Beijing and Guangzhou,China[J].Ecotoxicology,2016,25(3):439-446.
[5]ROBERTSON A M,BIEWENER A A.Muscle function during takeoff and landing flight in the pigeon(Columba livia)[J].J Exp Biol,2012,215(Pt23):4104-4114.
[6]MAURO A.Satellite cell of skeletal muscle fibers[J].JBiophys Biochem Cytol,1961,9(2):493-495.
[7]ASAKURA A,KOMAKI M,RUDNICKI M.Muscle satellite cells are multipotential stem cells that exhibit myogenic,osteogenic,and adipogenic differentiation[J].Differentiation,2001,68(4/5):245-253.
[8]吕捷,赵春礼,李玉成,等.成人骨骼肌细胞原代培养[J].神经解剖学杂志,2001,17(1):75-76.
[9]代阳,王轶敏,刘新峰,等.小鼠骨骼肌卫星细胞的分离培养和鉴定[J].天津农学院学报,2014(1):1-4.
[10]边艳超,牧仁,李向臣,等.鲁西黄牛胚胎骨骼肌卫星细胞分离培养及生物学特性[J].华中农业大学学报,2013,32(2):90-96.
[11]郑琪,睢梦华,朱龙,等.安淮山羊骨骼肌卫星细胞的分离培养与鉴定[J].安徽农业大学学报,2017,44(2):198-202.
[12]李方华,侯玲玲,马月辉,等.北京油鸡骨骼肌卫星细胞的分离、培养、鉴定及成肌诱导分化的研究[J].中国农业科学,2010,43(22):4725-4731.
[13]单艳菊,束婧婷,宋迟,等.鸭骨骼肌卫星细胞的分离培养与鉴定[J].江苏农业科学,2012,40(12):26-28.
[14]樊廷俊,徐晓辉,姜国建,等.条斑星鲽连续性鳍细胞系的建立与鉴定[J].山东大学学报(理学版),2010,45(5):22-27.
[15]周海波,蔡建庭,李茂岚,等.白藜芦醇诱导人胃癌原代细胞裸鼠移植瘤凋亡的研究[J].中国病理生理杂志,2005,21(3):528-532.
[16]庄鹏,江元森,马会慧,等.长期培养的大鼠原代肝细胞功能和形态学观察[J].中国病理生理杂志,2005,21(5):1001-1005.
[17]李方华,弓慧敏,冯士彬,等.骨骼肌卫星细胞的研究进展[J].中国畜牧兽医,2010,37(10):25-28.
[18]陈岩,王琨,朱大海.鸡骨骼肌卫星细胞的分离培养、鉴定及生物学特性研究[J].遗传,2006,28(3):257-260.
[19]夏家红,谢艾妮,徐磊,等.大鼠骨骼肌卫星细胞体外培养的实验研究[J].中华实验外科杂志,2005,22(2):214-215.
[20]TAKAYA T,NIHASHI Y,KOJIMA S,et al.Autonomous xenogenic cell fusion of murine and chick skeletal muscle myoblasts[J].Anim Sci J,2017,88(11):1880-1885.
[21]LAZARIDES E,HUBBARD B D.Immunological characterization of the subunit of the 100 A filaments from muscle cells[J].Proc Natl Acad Sci USA,1976,73(12):4344-4348.
[22]陈永乐,周光前,邓宇斌,等.C2C12成肌细胞体外诱导分化为肌管的实验[J].中山大学学报(医学科学版),2008,29(1):10-15.
[23]王静,宋新磊,崔焕先,等.骨骼肌卫星细胞的特性及其增殖与分化的调控[J].中国细胞生物学学报,2007,29(4):497-502.
[24]宋佳,曾缨,郑民缨,等.肌肉特异性微小RNA和成肌调节因子在C2C12细胞成肌分化过程中的表达[J].中国组织工程研究,2012,16(32):5999-6005.
[25]冯永强,李峥,褚万立,等.大鼠骨骼肌卫星细胞的原代培养鉴定和体外分化特点[J].中华医学杂志,2016,96(12):971-974.
[26]邱华玲,于建兴,陈宏权.MyoG基因结构与功能研究进展[J].生物信息学,2007,5(4):190-192.
[27]LING Y H,SUI M H,ZHENG Q,et al.miR-27b regulates myogenic proliferation and differentiation by targeting Pax3 in goat[J].Sci Rep,2018,8:3909.doi:10.1038/s41598-018-22262-4.