中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (49): 9146-9151.doi: 10.3969/j.issn.2095-4344.2012.49.005

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

Hoechst33342对大鼠骨髓间充质干细胞的标记

 

谢兴文1，侯费祎2，李 宁2，李盛华1，宋 敏2
  

  1. 1甘肃省中医药研究院，甘肃省兰州市 730050；2甘肃中医学院，甘肃省兰州市
    730000
  • 收稿日期:2012-01-10 修回日期:2012-03-19 出版日期:2012-12-02 发布日期:2012-03-19
  • 作者简介:谢兴文☆，男，1972 年生，甘肃省甘谷市人，2006 年上海中医药大学毕业，博士，副主任医师，硕士生导师，主要从事关节骨科和基础研究。xxw19726@hotmail.com
  • 基金资助:

    国家自然科学基金项目(81060299)。

Hoechst33342 for labeling rat bone marrow mesenchymal stem cells

Xie Xing-wen1, Hou Fei-yi2, Li Ning2, Li Sheng-hua1, Song Min2   

  1. 1Pharmaceutical Research Institute of Gansu Province, Lanzhou 730050, Gansu Province, China; 2Gansu University of Traditional Chinese Medicine, Lanzhou 730000, Gansu Province, China
  • Received:2012-01-10 Revised:2012-03-19 Online:2012-12-02 Published:2012-03-19
  • About author:Xie Xing-wen☆, Doctor, Associate chief physician, Master’s supervisor, Pharmaceutical Research Institute of Gansu Province, Lanzhou 730050, Gansu Province, China xxw19726@hotmail.com
  • Supported by:

    Supported by: the National Natural Science Foundation of China, No.81060299*

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774

被引次数

3

摘要：

背景：hoechst33342可以用于对骨髓间充质干细胞的标记，但是其标记的最佳浓度的研究目前尚未见报道。
目的：观察不同浓度的Hoechst33342对大骨髓间充质干细胞的增殖、标记率的影响，以及其荧光持续时间。
方法：贴壁筛选提取大鼠骨髓间充质干细胞，培养至第3代，流式细胞仪检测其表面抗原，成骨、成脂诱导后染色，用不同浓度Hoechst33342标记大鼠骨髓间充质干细胞后，检测标记后的大鼠骨髓间充质干细胞增殖率、标记率，荧光显微镜下观察其持续时间。
结果与结论：① hoechst33342可以用于骨髓间充质干细胞的标记，其最佳标记浓度为5 mg/L，标记持续时间较长，30 d未见猝灭。②其荧光激发后对细胞有损伤作用，标记浓度超过10 mg/L时，可致细胞死亡。③标记浓度在7.5 mg/L时，细胞增殖受到抑制；标记浓度在2.5 mg/L时，标记时间较短，7 d时荧光减弱，14 d荧光消失。结果表明，hoechst33342可用于骨髓间充质干细胞的标记，其对骨髓间充质干细胞标记的最佳浓度为5 mg/L；细胞表型鉴定CD45阴性，CD44，CD90均呈阳性表达。

关键词: 细胞标记, 骨髓间充质干细胞, Hoechst33342, 最佳浓度, 标记率, 干细胞

Abstract:

BACKGROUND: Hoechst33342 can be used for labeling bone marrow mesenchymal stem cells, but there are few reports on the optimal concentration of marker.
OBJECTIVE: To investigate the effect of different concentrations of Hoechst33342 on the proliferation and labeling rate of rat bone marrow mesenchymal stem cells and to investigate the duration time of fluorescence.
METHODS: Adherence screening method was used to extract rat bone marrow mesenchymal stem cells, and the cells were cultured to the third generation, then surface antigen was detected by flow cytometry, and stained after osteogenic and adipogenic induction. The proliferation rate and labeling rate of rat bone marrow mesenchymal stem cells were detected after treatment with different concentrations of Hoechst33342, and then the duration time was detected through the use of fluorescence microscope.
RESULTS AND CONCLUSION: Hoechst33342 can be used for labeling bone marrow mesenchymal stem cells, and the optimal concentration of Hoechst33342 was 5 mg/L, the duration time was long and there was no quenching within 30 days. Hoechst33342 damaged the cells after fluorescence excitation, and when the concentrations exceed10 mg/L, the marker could cause cell death. When the concentration was 7.5 mg/L, cell proliferation was inhibited; when the concentration was 2.5 mg/L, the duration for labeling was shorter, the fluorescence decreased at 7 days and disappeared at 14 days. Hoechst33342 can be used for labeling bone marrow mesenchymal stem cells, and the optimal concentration of the Hoechst33342 is 5 mg/L; phenotype identification shows the negative expression of CD45 and positive expression of CD44 and CD90.

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