中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (10): 1825-1828.doi: 10.3969/j.issn.1673-8225.2010.10.024

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

犬骨髓间充质干细胞片层的体外培养与制备

荆  恒，谭  帅，高振华，陈立强，李宁毅   

  1. 青岛大学医学院附属医院口腔颌面外科，山东省青岛市 266003
  • 出版日期:2010-03-05 发布日期:2010-03-05
  • 通讯作者: Li Ning-yi, Professor, Department of Oral and Maxillofacial Surgery, Hospital Affiliated to Medical College, Qingdao University, Qingdao 266003, Shandong Province, China ningyili342@163.com
  • 作者简介:荆 恒，男，1982年生，山东省淄博市人，汉族，青岛大学医学院在读硕士，主要从事骨组织工程学方面的研究。 jingheng1982@ 126.com
  • 基金资助:

    国家自然科学基金资助项目（30872896）；
    山东省自然科学基金资助项目（Y2008C77）。

Culture and preparation of dog bone marrow mesenchymal stem cell sheet in vitro

Jing Heng, Tan Shuai, Gao Zhen-hua, Chen Li-qiang, Li Ning-yi   

  1. Department of Oral and Maxillofacial Surgery, Hospital Affiliated to Medical College, Qingdao University, Qingdao   266003, Shandong Province, China
  • Online:2010-03-05 Published:2010-03-05
  • Contact: 李宁毅，教授，青岛大学医学院附属医院口腔颌面外科，山东省青岛市 266003 ningyili342@ 163.com
  • About author:Jing Heng, Studying for master’s degree, Department of Oral and Maxillofacial Surgery, Hospital Affiliated to Medical College, Qingdao University, Qingdao 266003, Shandong Province, China jingheng1982@126. com
  • Supported by:

    the National Natural Science Foundation of China, No. 30872896*;
    the Natural Science Foundation of Shandong Province, No. Y2008C77*

PDF

384

被引次数

12

摘要：

背景：传统的组织工程方法在收获和转移细胞方面存在诸多不足，且难以形成致密的组织，明显限制了组织工程学的发展。

目的：探讨犬骨髓间充质干细胞片层的体外培养与制备方法。

方法：实验犬麻醉后抽取骨髓，采用密度梯度离心法体外分离骨髓间充质干细胞。取传至第4代细胞，按1×109 L-1密度接种于直径3.5 cm的温度反应性培养皿中，置于37 ℃、体积分数为5%的CO2饱和湿度培养箱内培养，待细胞增殖融合时再将培养皿的温度降至20 ℃培养20 min，倒置显微镜下观察细胞形态学变化及细胞片层形成情况。

结果与结论：犬骨髓间充质干细胞原代培养24 h呈椭圆形或多角形，72 h多数细胞伸展贴壁，7 d时形成细胞集落，12 d时细胞呈长梭形，完全融合，细胞界限不清。温度反应性培养皿中的骨髓间充质干细胞在降温至20 ℃时，细胞形态发生变化，呈短梭形，且逐步从培养皿底分离，形成含细胞外基质的完整的细胞片层。证实密度梯度离心法可成功分离培养犬骨髓间充质干细胞，通过改变应用温度反应性培养皿的温度，能获取完整的细胞片层。

关键词: 温度反应性培养皿, 培养, 制备, 细胞片层, 骨组织工程, 骨髓间充质干细胞

Abstract:

BACKGROUND: There are some disadvantages in harvesting and transferring cells in the traditional tissue engineering technique, and it is difficult to form dense tissues, which significantly limits the development of tissue engineering.

OBJECTIVE: To explore the culture and fabrication of dog bone marrow mesenchymal stem cell (BMSC) sheet in vitro.

METHODS: Bone marrow was extracted from dogs following anesthesia. BMSCs were isolated with the method of density gradient centrifugation in vitro. BMSCs at passage 4 at a density of 1×109/L were incubated in the temperature-responsive culture dishes with a diameter of 3.5 cm, and cultured in an incubator at 37 ℃, 5% CO2 and saturated humidity. The temperature of the incubator was changed from to 37 ℃ to 20 ℃ to prepare BMSCs cell sheet for 20 minutes. Cell morphological changes and cell sheet formation were observed under an inverted microscope.

RESULTS AND CONCLUSION: Dog BMSCs following 24 hours of primary culture presented ellipse or polygonal shape. Most cells adhered at hour 72, and cell colonies were visible at day 7. Cells showed long spindle and completely confluence at day 12, with unclear boundary. BMSCs in the temperature-responsive culture dishes presented short spindle shape, and gradually separated from the dish bottom, forming entire cell sheet containing extracellular matrix at 20 ℃. These verified that dog BMSCs can be effectively obtained with method of density gradient centrifugation. Complete cell sheet layer can be fabricated with temperature-responsive culture dishes.

中图分类号: