中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (10): 1884-1888.doi: 10.3969/j.issn.1673-8225.2012.10.038

• 干细胞与中医药 stem cells and traditional Chinese medicine • 上一篇    下一篇

碱性成纤维细胞生长因子预诱导后加入丹参酮ⅡA体外定向培养骨髓间充质干细胞向神经元样细胞的分化***

李玉美1，余德立2，刘来兵3，余资江1   

  1. 1贵阳医学院人体解剖学教研室，贵州省贵阳市  550004；2贵阳中医学院临床二院系，贵州省贵阳市  550001；3贵阳医学院附属医院神经外科，贵州省贵阳市  550004
  • 收稿日期:2011-06-28 修回日期:2011-09-14 出版日期:2012-03-04 发布日期:2012-03-04
  • 通讯作者: 余资江，博士，教授，主要从事神经损伤、修复及再生研究。yuzijiang@yahoo.cn
  • 作者简介:李玉美，女，1983年生，四川省绵阳市人，汉族，2011年贵阳医学院毕业，助教，主要从事神经损伤、修复及再生研究。 136721361@qq.com
  • 基金资助:

    国家自然科学基金资助项目(30660189，81060108)；贵州省科技厅社会发展攻关项目(黔科合SY字[2009]3058)。

Tanshinone-ⅡA induced differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells in vitro after pre-induced by basic fibroblast growth factors

Li Yu-mei1, Yu De-li2, Liu Lai-bing3, Yu Zi-jiang1   

  1. 1Department of Anatomy, Guiyang Medical University, Guiyang  550004, Guizhou Province, China; 2Second Clinical Department, Guiyang University of Traditional Chinese Medicine, Guiyang  550001, Guizhou Province, China; 3Neurosurgery Department, Guiyang University of Traditional Chinese Medicine, Guiyang  550004, Guizhou Province, China
  • Received:2011-06-28 Revised:2011-09-14 Online:2012-03-04 Published:2012-03-04
  • Contact: author: Yu Zi-jiang, M.D., Professor, Department of Anatomy, Guiyang Medical University, Guiyang 550004, Guizhou Province, China yuzijiang@yahoo.cn
  • About author:Li Yu-mei, Teaching assistant, Department of Anatomy, Guiyang Medical University, Guiyang 550004, Guizhou Province, China 136721361@qq.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30660189*, 81060108*; Social Development Foundation of Science and Technology Department of Guizhou Province, No. SY[2009]3058*

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摘要：

背景：采用中药作为诱导剂对骨髓间充质干细胞向神经元样细胞定向诱导分化是否会有效果呢？
目的：验证碱性成纤维细胞生长因子预诱导后加入中药丹参酮ⅡA体外诱导骨髓间充质干细胞向神经元样细胞分化的效应。
方法：采用Percoll分离液密度梯度离心法获取骨髓间充质干细胞，取第3代细胞利用碱性成纤维细胞生长因子联合丹参酮ⅡA诱导其向神经元样细胞分化，全反式维甲酸无血清培养基诱导液及不进行诱导的细胞做为对照。
结果与结论：①骨髓间充质干细胞能稳定表达间质细胞特异性的标记物CD44，表达阳性率为(91.00±1.58)%，但不表达CD34、CD45，流式细胞学检测其纯度高达95.5%。②诱导分化后经扫描电镜检测可观察到典型的神经元样细胞结构；免疫组织化学检测细胞表达神经元特导性烯醇化酶，神经元特导性烯醇化酶阳性率为(75.60±2.31)%，Nestin呈一过性表达增加后随着诱导时间延长逐渐减低至阴性，不表达神经胶质纤维酸性蛋白。说明经碱性成纤维细胞生长因子预诱导后加入丹参酮ⅡA能高效地定向诱导神经元样细胞的分化。

关键词: 丹参酮ⅡA, 骨髓间充质干细胞, 神经元样细胞, 诱导分化, 差速贴壁

Abstract:

BACKGROUND: Whether bone marrow mesenchymal stem cells (BMSCs) induced by traditional Chinese medicine could differentiate into neuron-like cells remains poorly understood.                             
OBJECTIVE: To identify the efficiency of tanshinone-ⅡA containing traditional Chinese medicine on the differentiation of rat BMSCs into neuron-like cells in vitro after pre-induced by basic fibroblast growth factors.                                            
METHODS: One month old healthy male Wistar rats were used in this experiment, and BMSCs were separated and purified by Percoll medium density gradient centrifugation, repeated subculture and differential adherence methods. Passage 3 BMSCs were differentiated into neuron-like cells induced by basic fibroblast growth factors (bFGF) and tanshinone-ⅡA. All-trans retinoic acid serum-free medium induced cells and non-induced cells served as controls.                                            
RESULTS AND CONCLUSION: ①BMSCs could stably express the stem cells specific markers CD44, and the positive rate was (91.00±1.58)%. And the negative expressions of CD34 and CD45 were observed in the BMSCs by immunohistochemistry and flow cytomtry and the purity of stem cells was up to 95.5%. ② After directional differentiation and cultivation, typical neuron-like cells could be seen under scanning electron microscope. Positive expression of NSE was detected by immunohistochemistry and the possitive rate was (75.6±2.31)%. Nestin expression was gradually decreased to negative as well as the expression of glial fibrillary acidic protein. The results suggested that tanshinone-ⅡA can directly and efficiently induce BMSCs differentiating into neuron-like cells after pre-induced by basic fibroblast growth factors.
 

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