中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (19): 3421-3426.doi: 10.3969/j.issn.1673-8225.2010.19.001

• 干细胞培养与分化 stem cell culture and differentiation •    下一篇

BrdU标记大鼠骨髓间充质干细胞增殖与成骨能力的变化

冯进益，尤元璋，李  偏，蔡志刚，芮  钢   

  1. 福建医科大学附属厦门第一医院骨科，福建省厦门市  361003
  • 出版日期:2010-05-07 发布日期:2010-05-07
  • 通讯作者: 芮 钢，主任医师，硕士生导师，福建医科大学附属厦门第一医院骨科，福建省厦门市 361003 reigang@yahoo.com.cn
  • 作者简介:冯进益，男，1982年生，福建省永春县人，汉族，福建医科大学2007级在读硕士，医师，主要从事脊柱外科与组织工程的研究。 fjy10009@sina.com

Detection of changes in proliferation and osteogenic potentiality of rat bone marrow mesenchymal stem cells using BrdU labeling

Feng Jin-yi, You Yuan-zhang, Li Pian, Cai Zhi-gang, Rui Gang   

  1. Department of Orthopaedics, Xiamen First Hospital Affiliated to Fujian Medical University, Xiamen  361003, Fujian Province, China
  • Online:2010-05-07 Published:2010-05-07
  • Contact: Rui Gang, Chief physician, Master’s supervisor, Department of Orthopaedics, Xiamen First Hospital Affiliated to Fujian Medical University, Xiamen 361003, Fujian Province, China reigang@yahoo.com.cn
  • About author:Feng Jin-yi, Studying for master’s degree, Physician, Department of Orthopaedics, Xiamen First Hospital Affiliated to Fujian Medical University, Xiamen 361003, Fujian Province, China fjy10009@sina.com
  • Supported by:

    the Scientific Research Foundation of Health Bureau of Xiamen City, No. WSK0610*

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被引次数

3

摘要：

背景：BrdU标记是一种标记率高、标记简便的标记物，但其毒性报道不一。

目的：观察BrdU标记对SD大鼠骨髓间充质干细胞增殖与成骨能力的影响。

方法：直接贴壁法分离培养大鼠骨髓间充质干细胞，0.2%Brdu标记后检测其增殖能力、克隆形成能力；经成骨诱导液诱导培养3周后，利用茜素红染色，荧光免疫组织化学和RT-PCR观测标记前后骨髓间充质干细胞成骨能力的变化情况。

结果与结论：Brdu标记后骨髓间充质干细胞克隆形成能力明显减弱，在克隆个数和面积上均小于正常对照组(P < 0.05)；在增殖能力方面，BrdU标记后第4天开始进入对数增长期时增殖较慢，高峰较低(P < 0.05)；在成骨能力方面，经成骨诱导后BrdU标记组与正常对照组的骨髓间充质干细胞均表达骨钙素、Ⅰ型胶原，并且都有明显的钙结节形成，RT-PCR检测显示标记前后骨髓间充质干细胞在骨钙素基因的表达方面差异无显著性意义(P > 0.05)。提示BrdU标记抑制骨髓间充质干细胞的克隆增殖形成能力，但不会降低其成骨分化能力，可以广泛应用于骨髓间充质干细胞作为种子细胞干细胞研究的示踪剂。

关键词: 细胞示踪, BrdU, 骨髓间充质干细胞, 组织工程, Real Time-PCR

Abstract:

BACKGROUND: BrdU is a simple marker with high labeling rate, but there are various reports concerning its toxity.

OBJECTIVE: To observe the effects of BrdU labeling on proliferation and osteogenic potentiality of bone marrow mesenchymal stem cells (BMSCs) of Sprague Dawley rats.

METHODS: Rat BMSCs were isolated and cultured by direct adherent method. The ability of monoclone and proliferation was detected after labeling with 0.2% BrdU. Following 3 weeks of culture with osteogenic inducer, changes in osteogenic ability of BMSCs were observed using alizarin red staining, fluorescence immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) before and after labeling.

RESULTS AND CONCLUSION: The proliferation and monoclone of BMSCs were decreased obviously after BrdU labeling. The number and area of clones in BrdU labeling group were less than the normal control group (P < 0.05). The proliferation of the BrdU labeling group was slower when entering increased logarithmic phase at day 4, and the peak level was low (P < 0.05). Regarding osteogenic ability, BMSCs expressed osteocalcin, type Ⅰ collagen following osteogenic induction in the labeling and normal control group, with significant calcium nodules. However, the RT-PCR analysis showed that there was no significant difference in the expression of osteocalcin gene in BMSCs before and after labeling (P > 0.05). Above-mentioned results indicated that BrdU labeling inhibited proliferation and monoclone of BMSCs, but could not decrease its ability of osteogenic differentiation, and could be widely used as a tracer in research of BMSCs as seed cells.

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