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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
- 第三军医大学学报 2018年40卷第18期 页码：1630-1635
- 作者机构：
1.401121 重庆，重庆医科大学第四临床学院康复中心
2.400038 重庆，陆军军医大学(第三军医大学)药学与检验医学系临床血液学教研室
- 作者简介：
[ 舒彬，E-mail: Shubin1017@163.col SHU Bin, E-mail: Shubin1017@163.col
- 基金信息：国家自然科学基金面上项目(31571242)
DOI：10.16016/j.1000-5404.201803136
中图分类号： R322.74;R329-33;R329.2l收稿：2018-03-19，
修回：2018-05-31，
纸质出版：2018-09-30
- 稿件说明：
移动端阅览
卜凡, 解栋梁, 舒彬, 等. 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[J]. 第三军医大学学报, 2018,40(18):1630-1635.
Fan BU, Dongliang XIE, Bin SHU, et al. Fluorescence-activated cell sorting coupled with multiplex immunofluorescent labeling for assessing age-related changes in quantity and differentiation capability of mouse muscle satellite cells[J]. Journal of Third Military Medical University, 2018, 40(18): 1630-1635.
卜凡, 解栋梁, 舒彬, 等. 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[J]. 第三军医大学学报, 2018,40(18):1630-1635. DOI： 10.16016/j.1000-5404.201803136.
Fan BU, Dongliang XIE, Bin SHU, et al. Fluorescence-activated cell sorting coupled with multiplex immunofluorescent labeling for assessing age-related changes in quantity and differentiation capability of mouse muscle satellite cells[J]. Journal of Third Military Medical University, 2018, 40(18): 1630-1635. DOI： 10.16016/j.1000-5404.201803136.
摘要
目的
2
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
2
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
2
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(eMHC)，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
2
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。
Abstract
Objective
2
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
2
The hindlimb muscles including the gastrocnemius were harvested from male C57BL 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) and α7-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
2
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 (eMHC). 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
2
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.
关键词
Keywords
references
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