中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (10): 1764-1768.doi: 10.3969/j.issn.1673-8225.2011.10.012

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

克隆筛选纯化在大鼠脂肪源性干细胞原代培养中的作用

刘  剑1，2，李  立2，冉江华2，张升宁2，邵建春3   

  1. 1昆明医学院第二附属医院干疗外科，云南省昆明市650031；昆明医学院附属甘美医院，2肝胆胰外科， 3检验科，云南省昆明市  650011
  • 收稿日期:2010-11-28 修回日期:2011-01-18 出版日期:2011-03-05 发布日期:2011-03-05
  • 通讯作者: 李立，教授，主任医师，昆明医学院附属甘美医院肝胆胰外科，云南省昆明市650011 ynkmlili@yahoo.com
  • 作者简介:刘剑☆，男，1973年生，云南省昆明市人，汉族，昆明医学院在读博士，主治医师，主要从事肝胆外科基础及临床的研究。 Liujianwxc1219@163.com
  • 基金资助:

    云南省科技厅科研基金资助项目(2007CA007)“多脏器联合移植重大关键技术的研究与应用示范”。

Influence of monoclonal screening on the purification of primary cultured rat adipose tissue-derived stem cells

Liu Jian1, 2, Li Li2, Ran Jiang-hua2, Zhang Sheng-ning2, Shao Jian-chun3   

  1. 1Depatrment of Surgery, Second Affiliated Hospital of Kunming Medical University, Kunming  650031, Yunnan Province, China; 2Department of Hepatobiliary and Pancreatic Surgery, Affiliated Ganmei Hospital of Kunming Medical University, Kunming  650011, Yunnan Province, China; 3Department of Laboratory Medicine, Affiliated Ganmei Hospital of Kunming Medical University, Kunming  650011, Yunnan Province, China
  • Received:2010-11-28 Revised:2011-01-18 Online:2011-03-05 Published:2011-03-05
  • Contact: Li Li, Professor, Chief physician, Department of Hepatobiliary and Pancreatic Surgery, Affiliated Ganmei Hospital of Kunming Medical University, Kunming 650011, Yunnan Province, China ynkmlili@yahoo.com
  • About author:Liu Jian☆, Studying for doctorate, Attending physician, Department of Surgery, Second Aftiliated Hospital, Kunming Medical University, Kunming 650031, Yunnan Province, China; Department of Hepatobiliary and Pancreatic Surgery, Affiliated Ganmei Hospital of Kunming Medical University, Kunming 650011, Yunnan Province, China Liujianwxc1219@163.com
  • Supported by:

    the Foundation of Science and Technology Department of Yunnan Province, No. 2007CA007*

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摘要：

背景：研究中发现从成体的脂肪组织中可以分离出一种间质干细胞，即脂肪源性干细胞，但传统的分离方法仍存在一些问题需要改进。
目的：在脂肪源性干细胞传统的分离方法的基础上进行改进，克隆筛选纯化脂肪源性干细胞。
方法：消化离心法分离Lewis大鼠脂肪源性干细胞，有限密度稀释法克隆化及条件培养基筛选，进行原代培养，以克隆筛选的脂肪源性干细胞为实验组，未克隆筛选的脂肪源性干细胞为对照组，观察细胞形态，对比生长曲线、倍增时间，流式细胞仪鉴定脂肪源性干细胞表面标记CD49d、CD29、CD44。
结果与结论：经克隆筛选的脂肪源性干细胞与未经克隆筛选的脂肪源性干细胞相比，前者形态均一，生长力旺盛，倍增时间短，多代培养后生长曲线无明显改变，通过流式细胞仪鉴定，实验组与对照组在3~6代之间均表达表面标记CD49d、CD29、CD44。结果证实克隆筛选的纯化方法是脂肪源性干细胞原代培养的有效、经济的纯化方法。

关键词: 脂肪源性干细胞, 克隆筛选, 原代培养, 大鼠, 流式细胞仪

Abstract:

BACKGROUND: It was found that a kind of mesenchymal stem cell can be isolated from animal adult adipose tissues, that is, adipose tissue-derived stem cells (ADSCs). However, there were still some problems need to be improved of the traditional isolation methods.
OBJECTIVE: To investigate a new method that using monoclonal screening in the purification of primary cultured ADSCs. 
METHODS: After isolated the Lewis rat ADSCs with the digestion centrifuge process, the ADSCs were screened with the limited density dilution cloning and condition culture medium screening in the original generation culture. The group with clone screening ADSCs served as the experimental group, without clone screening as the control group. Cellular appearance, growth curve and cell doubling generation time were compared between groups. The surface markers CD49d, CD29, CD44 of ADSCs were determined by flow cytometry.
RESULTS AND CONCLUSION: The appearance of ADSCs which underwent clone screening had been homogeneously, with vigorous growing power and short doubling generation time. The growth curve was not changed obviously after many generations. The surface marks CD49d, CD29, and CD44 were expressed in both groups at 3-6 passages. The clone screening purification method is effective, economical purification method of ADSCs.

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