山羊骨骼肌卫星细胞系分离、培养及其成肌诱导分化研究  

Isolation,culture and differentiation of goat skeletal satellite cell

作　　者：李伟[1] 郑蒙蒙[1] 邱峰龙[1] 朱才业[1] 韦光辉[1] 王丹[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2013年第1期12-17,共6页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：优质转基因肉羊新品种培育项目(2011ZX08008-003);国家高技术研究发展计划项目(863-2011AA100307-04)

摘　　要：采用外科手术方法从山羊四肢肌肉或背最长肌获取3mm×3mm肌肉束,置于1mg.mL-1 IV胶原酶中,室温消化30min,分散肌丝,在体视显微镜下分离肌丝;再用0.25%胰酶-EDTA于37℃下消化10min,以释放出骨骼肌卫星细胞(SSC)和其他细胞。连续5代在增殖培养体系(80%DMEM/F12+10%胎牛血清+10%马血清)中采用差速贴壁方法,收集接种后20min的贴壁细胞,对纯化的SSC采用免疫荧光方法检测SSC特异标志蛋白Pax-7,结果表明:(92.5±3.4)%细胞表达Pax-7,(93.0±1.8)%细胞表达MyoD,说明90%以上细胞为SSC。将纯化的SSC细胞分别在诱导培养体系Ⅰ(93%DMEM/F12+1%胎牛血清+1%马血清+1%non-AA)和II(99%Opti-MEM+1%non-AA)中进行诱导培养,通过细胞形态学观察和成肌细胞早期标志蛋白Desmin、MyHC免疫荧光检测诱导效果,结果表明:采用体系Ⅱ能在各种细胞密度(大于1×105个.cm-2)下诱导山羊SSC向成肌方向分化,而采用体系Ⅰ(低血清培养基)且仅当能诱导高密度山羊SSC向成肌方向分化。采用改进的SSC分离方法能够获得较高纯度的卫星细胞,比较诱导体系Ⅰ、Ⅱ认为,诱导培养体系Ⅱ适合于山羊SSC成肌诱导分化,为进一步研究SSC成肌分化机制提供素材。The paper was to provide a stable isolation method of skeletal satellite cell and a suitable media for its differ- entiation. For the specific cell localization, which located between the basement-and cytoplasm-membrane, the myofibers were separated under the style microscope after being loosen by collagenase type IV at 1 mg ·mL-1 for about 30 min. Af- ter washed with PBS, the separated myofihres were digested by 0.25 % trypsin-EDTA to release cells in the myofibres. Then the cells suspension were incubated at 37 ℃, 5% CO2. At last satellite cell was cultured in proliferation media （80% DMEM/F12＋10% FBS＋10% HS） and purified by the difference of the adhension time. After the 6th passage, the cell was identified by the indirect immunofluorescence assay （IFA）. Differentiantion media I （93% DMEM/F12 ＋ 1% FBS＋1% HS＋1% non-AA） and differentiantion media Ⅱ（99% Opti-MEM＋1% non-AA） were used to find a suitable media for the differentiation. The differentiation of satellite cell, cultured in differentiantion media Ⅱ , would dif- ferented at any cell density （〉1 × 10^5/cm2 ）, while the differentiantion media I only induced goat SSC at a high cell densi- ty. The IFA and differentiation rusults indicated skeletal satellite cell isolation method would obtain skeletal satellite cells with a high purity of~ 90% [Pax-7 pastive ratio was （92.5±3.4） %, and the MyoD pastive ratio was （93.0±1.8） %]. Desmin and MyHC pastive ratio of induced goat SSC at 24 h was （92.3 ±1.1）% and （93.0± 1.2）% respectively. The purity of goat SSC was relatively high by the improved seperation method. Considering the serum concentration of prolif- eration and differentiation media I and the inducing ablity of differentiation media I and II , the differentiation media II was suitable for inducing the goat SSC. Furthermore the differentiation media II was serum-free media without any sub- stance of uncertain, which could make the study of Myo-differentiation easier.

关 键 词：山羊 骨骼肌卫星细胞 细胞培养 成肌诱导分化 

分 类 号：Q785[生物学—分子生物学]