Chinese Journal of Tissue Engineering Research ›› 2015, Vol. 19 ›› Issue (19): 2965-2972.doi: 10.3969/j.issn.2095-4344.2015.19.003

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Neuron-like differentiation of bone marrow mesenchymal stem cells induced by Rehmannia decoction with Cimicifuga heracleifolia Kom. and Achyranthes bidentata Bl. as guiding drugs 

Wu Mi-shan1, Zhao Su-zhi2, Gao Wei-juan3, Ren Li-zhong4, Wang Ru1, Liu Ying1, Han Hong-wei1, Li Bin5   

  1. 1Department of Formulaology, Basic Medicine College, Hebei University of Chinese Medicine, Shijiazhuang 050200, Hebei Province, China; 2Changan District Shengbei Community Health Center of Shijiazhuang, Shijiazhuang 050041, Hebei Province, China; 3Hebei Key Laboratory of Chinese Medicine Research on Cardio-Cerebrovascular Disease, Shijiazhuang 050091, Hebei Province, China; 4Department of Orthopedics, the First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei Province, China; 5Department of Biochemistry, Hebei Medical University, Shijiazhuang 050017, Hebei Province, China  
  • Online:2015-05-06 Published:2015-05-06
  • Contact: Corresponding author: Zhao Su-zhi, Associate chief physician, Changan District Shengbei Community Health Center of Shijiazhuang, Shijiazhuang 050041, Hebei Province, China
  • About author:Wu Mi-shan, M.D., Professor, Master’s supervisor, Department of Formulaology, Basic Medicine College, Hebei University of Chinese Medicine, Shijiazhuang 050200, Hebei Province, China
  • Supported by:

     the National Natural Science Foundation of China, No. 81073074, 30472200; the Natural Science Foundation of Hebei Province, No. H2013206005; the Major Scientific Research Project of Hebei Provincial Higher Education, No. ZD2015053; High-Level Personnel Scientific Research Projects of Higher Education in Hebei Province, No. GCC2014013

Abstract:

BACKGROUND: Rehmannia decoction has remarkable effects on the neurologic function recovery of cerebral ischemia-reperfusion model rats undergoing bone marrow mesenchymal stem cell transplantation.
OBJECTIVE: To investigate the feasibility of bone marrow mesenchymal stem cells differentiating into neuron-like cells induced by Rehmannia decoction with Cimicifuga heracleifolia Kom. and Achyranthes bidentata Bl. as guiding drugs and to further explore the regulatory role.
METHODS: Bone marrow mesenchymal stem cells were harvested from 4-week-old Sprague Dawley rats and cultured in vitro using the whole bone marrow adhesion method. Flow cytometry assay was used to detect the expression of CD90 and CD45 of bone marrow mesenchymal stem cells at passage 3. Cells at passage 3 were divided into four groups according to different culture conditions: blank control group (1% fetal bovine serum+DMEM/F-12), Rehmannia decoction group (serum containing Rehmannia decoction+1% fetal bovine serum+DMEM/F-12), Achyranthes bidentata Bl. group (serum containing Rehmannia decoction with Achyranthes bidentata Bl. as the guiding drug+1% fetal bovine serum+DMEM/F-12), Cimicifuga heracleifolia Kom. group (serum containing Rehmannia decoction with Cimicifuga heracleifolia Kom. as guiding drug+1% fetal bovine serum+DMEM/F-12). Then the cellular morphology was observed under inverted phase contrast microscope. At 7 days after induction, immunocytochemistry staining and western blot assay were used to detect expression of neuron-specific enolase and glial fibrillary acidic protein; and RT-PCR was used to detect mRNA expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells roughly presented a single long spindle or flat-shaped morphology at passage 3, grew with close whirlpool-like arrangement, and the CD90 expression level was up to 98%, but the expression level of CD45 was only 1.3%. At 7 days after induction, bone marrow mesenchymal stem cells in the groups of Rehmannia decoction, Achyranthes bidentata Bl. and Cimicifuga heracleifolia Kom. became round, extended multiple distinct processes and had connections among some processes, exhibiting typical neuron-like cell morphology, but in the blank control group, there was no obvious change. Immunocytochemistry staining showed that compared with the Rehmannia decoction group, the protein expression levels of neuron-specific enolase and glial fibrillary acidic protein in Achyranthes bidentata Bl. and Cimicifuga heracleifolia Kom. groups were obviously increased (P < 0.05); and these protein levels were also higher in the Cimicifuga heracleifolia Kom. group than the Achyranthes bidentata Bl. group (P < 0.05). Western blot results were consistent with the findings of immunocytochemistry staining. RT-PCR results showed the mRNA levels of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor were ranked as follows: Cimicifuga heracleifolia Kom. group < Achyranthes bidentata Bl. < Rehmannia decoction < blank control group, and there were significant differences between different groups (P < 0.05). Taken together, Rehmannia decoction with Cimicifuga heracleifolia Kom. and Achyranthes bidentata Bl. as guiding drugs can induce the neuronal differentiation of bone marrow mesenchymal stem cells, and Wnt/β-catenin signaling pathway may play an important role in neural differentiation of bone marrow mesenchymal stem cells.

Key words: Bone Marrow, Mesenchymal Stem Cells, Cimicifuga, Cell Differentiation

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