中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (6): 1072-1077.doi: 10.3969/j.issn.1673-8225.2010.06.025

• 干细胞与中医药 • 上一篇    下一篇

川芎嗪诱导大鼠骨髓间充质干细胞分化为神经元样细胞：最佳诱导剂量筛选

陈 兵1，尹延庆1，柯俊龙2，邹新辉1，彭 浩1，谈山峰1，许志恩2   

  1. 1广东医学院附属医院，1神经外科，2神经内科，广东省湛江市  524001
  • 出版日期:2010-02-05 发布日期:2010-02-05
  • 作者简介:陈 兵，男，1970年生，湖南省湘乡市人，汉族，2002年中南大学毕业，博士，副教授，主要从事创伤性脑损伤的研究。 drcb@163.com

Ligustrazine induces rat bone morrow mesenchymal stem cells to differentiate into neuron-like cells: Screening of the optimal inductive concentration

Chen Bing1, Yin Yan-qing1, Ke Jun-long2, Zou Xin-hui1, Peng Hao1, Tan Shan-feng1, Xu Zhi-en2   

  1. 1Department of Neurosurgery, 2Department of Neurology, Affiliated Hospital of Guangdong Medical College, Zhanjiang  524001, Guangdong Province, China
  • Online:2010-02-05 Published:2010-02-05
  • About author:Chen Bing, Doctor, Associate professor, Department of Neurosurgery, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, Guangdong Province, China drcb@163.com

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摘要：

背景：目前用于体外诱导骨髓间充质干细胞向神经元样细胞分化的诱导剂众多，但多数化学诱导剂具有毒性不适合用于人体。

目的：观察中药川芎嗪对大鼠骨髓间充质干细胞分化为神经元样细胞的影响，并寻找川芎嗪诱导分化的最佳浓度。

方法：SD大鼠麻醉后无菌条件下取出股骨和胫骨，离心后弃上清液，加入含体积分数为15%胎牛血清的L-DMEM培养基重新悬浮细胞并转入培养瓶培养传代，用免疫细胞化学方法检测第5代骨髓间充质干细胞CD44、CD45的表达；取含1.00，1.25，1.50 g/L 3种剂量盐酸川芎嗪注射液的无血清L-DMEM培养基对体外培养的第5代骨髓间充质干细胞进行诱导。倒置相差显微镜下观察细胞形态变化，免疫细胞化学方法检测已诱导细胞巢蛋白、神经元特异性烯醇化酶和胶质纤维酸性蛋白的表达，比较3种剂量盐酸川芎嗪注射液诱导神经元样细胞抗原表达率。

结果与结论：①原代细胞接种3 d后多数细胞贴壁，传代后细胞贴壁速度和增殖更快，第5代基本纯化为骨髓间充质干细胞，细胞呈放射状或漩涡状排列。②第5代骨髓间充质干细胞(98.02±0.81)%CD44表达阳性，CD45表达阴性。③诱导后细胞出现类似神经元细胞样形态；免疫细胞化学方法检测显示多数细胞巢蛋白、神经元特异性烯醇化酶阳性表达，1.25 g/L浓度组细胞的神经元特异性烯醇化酶阳性表达率最高。提示川芎嗪可诱导骨髓间充质干细胞分化为神经元样细胞，1.25 g/L为最适诱导剂量。

关键词: 川芎嗪, 骨髓间充质干细胞, 神经元样细胞, 诱导剂, 分化

Abstract:

BACKGROUND: There are numerous inducers used in inducing bone marrow mesenchymal stem cells (BMMSCs) differentiate into neuron-like cells, however, due to poisonous, most chemical inducers can not be used in human.

OBJECTIVE: To investigate the effect of ligustrazine on differentiation of rat BMMSCs into neuron-like cells in vitro, and to search for the optimal inductive concentration.

METHODS: After SD rats were anesthetized, bone marrow was obtained from the femoral and tibial bones, centrifuged, and the supernatant was discarded. The extracted cells were cultured in L-DMEM supplemented with 15% fetal bovine serum. The expression of CD44 and CD45 of the 5th passage of BMMSCs were identified by immunocytochemical technique. Serum-free L-DMEM medium contains 1.00, 1.25 and 1.50 g/L ligustrazine concentrations were used to induce the 5th passage of BMMSCs in vitro. Morphology changes of BMMSCs were observed under an inverted phase microscope. Expression of nestin, neuron-specific enolase and glial fibrillary acidic protein were identified by immunocytochemical technique, and the expression ratio of neuron-like cells’ surface antigens induced by different concentrations of ligustrazine were compared. 

RESULTS AND CONCLUSION: ①Most primarily cultured BMMSCs adhered to the wall at 3 days after culture, which proliferated faster after passaged, and the 5th passage of cells were mostly purified into BMMSCs, spread radially or vortex-likely. ②The 5th passage of BMMSCs was positive expressed (98.02±0.81)% CD44, but negative for CD45. ③Neuron-like cells with prominence and bifurcation could be seen after induction. The immunocytochemical method showed that nestin and neuron-specific enolase in most induced cells were positive expressed, especially received a highest ration of neuron-specific enolase expressing in the induced group with 1.25 g/L concentration of ligustrazine. It revealed that ligustrazine can induce BMMSCs differentiated into neuron-like cells, and 1.25 g/L is the optimal inductive concentration.

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