中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (36): 6797-6801.doi: 10.3969/j.issn.1673-8225.2011.36.034

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

应用改良差速贴壁法和有限稀释技术分离培养小鼠来源肌源干细胞

丁维进1，2，3，唐  乙1，2，宋艳玲1，2，苏志达2，李  翠2，刘安堂1，2，胡  霞3，江  华1，2   

  1. 解放军第二军医大学长征医院，1整形外科，2神经科学研究中心，上海市  200003；3解放军第264医院美容整形科，山西省太原市030001
  • 收稿日期:2011-04-12 修回日期:2011-08-01 出版日期:2011-09-03 发布日期:2011-09-03
  • 通讯作者: 江华，教授，主任医师，解放军第二军医大学长征医院， 整形外科，神经科学研究中心，上海市 200003 dosjh@126.com
  • 作者简介:丁维进☆，男，1982年生，江苏省海安县人，汉族，2011年解放军第二军医大学毕业，博士，主要从事周围神经损伤后修复的研究。 dingweijin@yahoo.cn
  • 基金资助:

    上海市基础研究重点课题(08JC1407100)。

Isolation of murine muscle-derived stem cells with preplate technique combined with limited dilution technique

Ding Wei-jin1,2,3, Tang Yi1,2, Song Yan-ling1,2, Su Zhi-da2, Li Cui2, Liu An-tang1,2, Hu Xia3, Jiang Hua1,2   

  1. 1Plastic and Reconstructive Department of Changzheng Hospital, Second Military Medical University, Shanghai  200003, China; 2Neuroscience Research Center of Changzheng Hospital, Second Military Medical University, Shanghai  200433, China; 3Esthetic and Plastic Surgery Department, the 264 Hospital of Chinese PLA, Taiyuan  030001, Shanxi Province, China
  • Received:2011-04-12 Revised:2011-08-01 Online:2011-09-03 Published:2011-09-03
  • Contact: Jiang Hua, Professor, Chief physician, Plastic and Reconstructive Department of Changzheng Hospital, Second Military Medical University, Shanghai 200003, China; Neuroscience Research Center of Changzheng Hospital, Second Military Medical University, Shanghai 200433, China dosjh@126.com
  • About author:Ding Wei-jin☆, Doctor, Plastic and Reconstructive Department of Changzheng Hospital, Second Military Medical University, Shanghai 200003, China; Neuroscience Research Center of Changzheng Hospital, Second Military Medical University, Shanghai 200433, China; Esthetic and Plastic Surgery Department, the 264 Hospital of Chinese PLA, Taiyuan 030001, Shanxi Province, China dingweijin@yahoo.cn
  • Supported by:

    the Basic Research Program of Shanghai, No. 08JC1407100*

PDF

680

被引次数

8

摘要：

背景：研究者们通过多种方法从肌组织中分离得到肌源干细胞，并应用于各类组织工程和再生医学研究。
目的：结合改良的差速贴壁法和有限稀释技术分离小鼠来源肌源干细胞，并培养其单细胞克隆和亚克隆集落。
方法：以新生C57BL/6小鼠四肢作为肌组织取材对象，经三重酶消化和细胞筛过滤，运用改良的差速贴壁法分离出肌源干细胞，予细胞特异标记物以免疫组织化学染色；以有限稀释技术克隆培养的方法，获得稳定的肌源干细胞单克隆和亚克隆集落。
结果与结论：差速贴壁培养过程中，肌性细胞占比逐渐增高，首次贴壁1 h可以获得足够数量的细胞进行第6次贴壁培养；肌源干细胞需72 h左右贴壁生长，经10 d左右可以增殖为300~500细胞数量的集落，细胞形态以小圆形细胞为主，并有少量梭形细胞，肌源干细胞能够维持形态并持续增殖；应用有限稀释技术可获得肌源干细胞单克隆和亚克隆集落，肌源干细胞克隆细胞均呈现Desmin染色阳性，Sca-1染色阳性，阳性率为(92.3±4.1)%。提示应用preplate法和有限稀释技术可以分离得到小鼠来源肌源干细胞及其克隆集落。

关键词: 肌源干细胞, 差速贴壁法, 有限稀释技术, 克隆, 亚克隆

Abstract:

BACKGROUND: Muscle-derived stem cells (MDSCs), a newly discovered adult stem cell possessing utility potential in tissue engineering and regenerative medicine, have been isolated from skeletal muscle tissue. The MDSCs isolating method varies a lot.
OBJECTIVE: To isolate and culture MDSCs and its clone as well as sub-clone through the use of modified preplate technique combined with limited dilution technique.
METHODS: The muscle tissue was obtained from new born mice under microscopy and then digested and filtered, from which MDSCs were isolated by modified preplate technique with first round plating period of 1 hour. The muscle derived cells were counted and MDSCs were marked with immunohistochemistry method. The MDSC clone and its subclone were obtained with limited dilution technique.
RESULTS AND CONCLUSION: During the isolation procedure with preplate technique, muscle derived cells made up a progressively higher ratio in the cell culture and the procedure with first round plating period of 1 hour provided plenty cells for MDSCs isolation. MDSCs presented with adherent growth 72 hours after the sixth suspension, grew into cell population of 300-500 cells in 10 days about, and proliferated with its small round and spindle morphology persisted. MDSCs clone and sub-clone were obtained through limited dilution technique and found desmin positively expressed and Sca-1 positively expressed at ratio of (92.3±4.1)%. The MDSCs and its clone from mice may provide proper cell resource for tissue engineering and regenerative medicine research.

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