中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (32): 5946-5952.doi: 10.3969/j.issn.2095-4344.2012.32.011

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

人脂肪干细胞的分离培养与生物学特性

田 霖，孙筱放，刘海波，骆玉梅，陈欣洁，黄东健   

  1. 广州医学院第三附属医院，广东省广州市 510150
  • 收稿日期:2012-03-05 修回日期:2012-04-27 出版日期:2012-08-05 发布日期:2012-08-05
  • 通讯作者: 陈欣洁，博士，主任技师，硕士生导师，广州医学院第三附属医院，广东省广州市 510150 并列通讯作者：黄东健，博士，主任医师，硕士生导师，广州医学院第三附属医院，广东省广州市 510150
  • 作者简介:田霖★，女，1984年生，湖北省宜昌市人，汉族，广州医学院在读硕士，主要从事干细胞与神经系统疾病研究。 doctor_tian118@yahoo.cn

Isolation, culture and biological characteristics of human adipose-derived stem cells in vitro

Tian Lin, Sun Xiao-Fang, Liu Hai-Bo, Luo Yu-Mei, Chen Xin-Jie , Huang Dong-Jian   

  1. Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China
  • Received:2012-03-05 Revised:2012-04-27 Online:2012-08-05 Published:2012-08-05
  • Contact: Chen Xin-jie, M.D., Chief technician, Master’s supervisor, Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China Co-corresponding author: Huang Dong-jian, M.D., Chief physician, Master’s supervisor, Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China
  • About author:Tian Lin★, Studying for master’s degree, Third Affiliated Hospital, Guangzhou Medical University, Guangzhou 510150, Guangdong Province, China doctor_tian118@yahoo.cn

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1188

被引次数

26

摘要：

背景：高效、稳定地获得大量较高纯度的人脂肪干细胞，是其在组织工程学及再生医学中广泛应用的基础和前提。
目的：探索体外培养脂肪干细胞的适宜条件，从而提高其增殖能力。
方法：采用胶原酶消化的方法，从人腹部皮下块状脂肪中分离出脂肪干细胞，经贴壁筛选法纯化细胞、低糖培养基体外培养扩增。Giesam染色后观察细胞形态；绘制细胞生长曲线并进行细胞周期分析，观察分析传代后染色体核型改变；选取第3代脂肪干细胞做流式细胞鉴定、EdU细胞增殖能力检测以及克隆形成实验。
结果与结论：分离培养的原代脂肪干细胞形态不一，经传代后的细胞形态趋于长梭形，排列紧密呈漩涡状生长。细胞生长曲线呈S形，细胞周期分析及EdU掺入法结果显示体外培养的脂肪干细胞具有较强的增殖能力。经染色体核型分析结果提示，体外培养不会引起脂肪干细胞的核型改变。第3代脂肪干细胞经流式细胞仪检测CD29，CD44，CD90，CD105均呈阳性表达，而CD34和CD45呈阴性；克隆形成率为8.8%；在一定的诱导条件下，脂肪干细胞能够向脂肪细胞及成骨细胞分化。结果说明采用胶原酶消化法可成功分离培养人脂肪干细胞，且具有较强的增殖能力。

关键词: 人脂肪干细胞, 分离, 体外扩增培养, 鉴定, 染色体核型, 生物学特性

Abstract:

BACKGROUND: Human adipose-derived stem cells are widely used as seed cells in tissue engineering and regenerative medicine. Therefore, harvesting a large number of high-purity human adipose-derived stem cells is important for above-mentioned studies.
OBJECTIVE: To explore the suitable culture condition of human adipose-derived stem cells in vitro, and to enhance the proliferative ability of human adipose-derived stem cells.
METHODS: Collagenase I was used to digest and isolate human adipose-derived stem cells from intact fat of human abdomen. Human adipose-derived stem cells were purified using attachment method, cultured with low-glucose medium and passaged in vitro. Morphology was observed after Giemsa staining; the growth curve was drawn and cell cycle was analyzed. The karyotype of the passaged human adipose-derived stem cells was analyzed. Cells at the third passage were subjected to flow cytometry analysis, EdU incorporation and colony forming experiments.
RESULTS AND CONCLUSION: The morphology of primary cultured human adipose-derived stem cells was not the same, passaged human adipose-derived stem cells were spindle-shaped and arranged tightly in a vortex-like appearance. The growth curve was “S” shaped. Cell cycle analysis and EdU incorporation results showed that human adipose-derived stem cells cultured in vitro could maintain strong proliferative ability. Karyotype mapping showed that in vitro culture could not cause chromosome abnormalities of adipose-derived stem cells. Flow cytometry analysis showed that passage 3 adipose-derived stem cells were positive for CD29, CD44, CD90 and CD105, but they were negative for CD34 and CD45, with a clone formation rate of 8.8%. Under certain induction condition, adipose-derived stem cells could differentiate into adipocytes and osteoblasts. Human adipose-derived stem cells were successfully isolated by collagenase digestion method, cultured in vitro and showed strong proliferative ability.

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