应用多重荧光标记的流式细胞分选技术检测小鼠肌卫星细胞数量及其分化能力的增龄性变化

Fluorescence-activated cell sorting coupled with multiplex immunofluorescent labeling for assessing age-related changes in quantity and differentiation capability of mouse muscle satellite cells

目的应用流式细胞分选技术(fluorescence-activated cell sorting,FACS)分离检测不同鼠龄小鼠骨骼肌卫星细胞数量并观察其成肌分化能力。方法取雄性C57BL小鼠腓肠肌等组织,机械剪碎后胶原酶/分散酶联合消化,过滤离散细胞进行免疫标记染色,流式细胞仪分选CD45-/CD31-/Sca1-/ɑ7-integrin+细胞群体,培养分选细胞并进行Pax7和Desmin肌原性分子鉴定和分化诱导;观察比较青年、成年和老年小鼠骨骼肌组织中静止肌卫星细胞数量和成肌分化能力的变化。结果分选出的成年CD45-/CD31-/Sca1-/α7-integrin+细胞群体约占离散细胞总数的2%~3%,呈现Pax7、Desmin等染色阳性,经分化诱导能形成典型肌管并表达胚胎肌球蛋白重链(e MHC),青年组小鼠静止卫星细胞比例及每克肌肉内肌卫星细胞数量明显较高(P<0.05),老年组则明显下降(P<0.05),同时其肌管融合指数及大肌管比例明显减少(P<0.05)。结论基于多重免疫荧光标记的流式分选术能实现骨骼肌卫星细胞的快速高效分离,分选细胞可诱导形成肌管;老年骨骼肌组织肌卫星细胞数量与功能呈现显著下降。

Objective To isolate and count of skeletal muscle satellite cells（MSCs） in mice at different ages based on fluorescence-activated cell sorting（FACS） coupled with multiplex immunofluorescent labeling and observe the differentiation capacity of the cells. Methods The hindlimb muscles including the gastrocnemius were harvested from male C57 BL mice and then cutted and digested with collagenase and dispase. The discrete cells were filtered and stained for CD45（FITC）,CD31（FITC）,Sca1（PE-cy7） andα7-integrin（PE）. The population of CD45-/CD31-/Sca1-/α7-integrin＋cells was sorted by flow cytometry,and after characterization for expression of the myogenic molecules Pax7 and Desmin,the sorted cells were cultured and induced for myogenic differentiation. The quantity and differentiation capacity of the MSCs from young,adult and aged mice were assessed. Results The population of CD45-/CD31-/Sca1-/α7-integrin＋cells,which were positive for Pax7 and Desmin,accounted for 2% - 3% in the total adult discrete cells. After myogenic differentiation,the cells formed typical myotubes and expressed embryonic myosin heavy chain（e MHC）. The total number of quiscent MSCs sorted and the number of quiscent MSCs per gram muscle were the largest in young mice（P 0. 05）,and were decreased significantly in aged mice（P 0. 05）. The myotube fusion index and the ratio of large myotube formation were significantly reduced in the MSCs derived from aged mice（P 0. 05）. Conclusion FACS coupled with multiplex immunofluorescent labeling allows efficient separation of mouse MSCs,which can form typical myotubes in culture. The number and differentiation capacity of quiscent MSCs in the skeletal muscles decrease significantly in aged mice.