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

doi:10.3969/j.issn.2095-4344.2015.19.003

Abstract

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

CLC Number:

R394.2

Wu Mi-shan1, Zhao Su-zhi, Gao Wei-juan, Ren Li-zhong, Wang Ru, Liu Ying, Han Hong-wei, Li Bin. Neuron-like differentiation of bone marrow mesenchymal stem cells induced by Rehmannia decoction with Cimicifuga heracleifolia Kom. and Achyranthes bidentata Bl. as guiding drugs [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(19): 2965-2972.

Figures/Tables 5

References

[1] Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418(6893):41-49.
[2] Ma K, Fox L, Shi G, et al. Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells. Neurol Res. 2011;33(10):1083-1093.
[3] Shi M, Liu ZW, Wang FS. Immunomodulatory properties and therapeutic application of mesenchymal stem cells. Clin Exp Immunol. 2011;164(1):1-8.
[4] Jiang J, Lv Z, Gu Y, et al. Adult rat mesenchymal stem cells differentiate into neuronal-like phenotype and express a variety of neuro-regulatory molecules in vitro. Neurosci Res. 2010;66(1):46-52.
[5] 武密山,李恩,赵素芝,等.补肾方含药血清对大鼠成骨细胞雌激素受体mRNA及其蛋白表达的影响[J].中国药理学通报,2008, 24(10):1396-1397.
[6] 茅晓.玄府气液学说及其临床应用[J].南京中医药大学学报,2000, 16(4):238-239.
[7] 李其忠.气门、玄府、腠理、三焦联考[J].上海中医药杂志,1998, 32(3):2-3.
[8] 郑国庆,黄培新.玄府与微循环和离子通道[J].中国中医基础医学杂志,2003,9(4):13-14.
[9] Zhu H, Guo ZK, Jiang XX, et al. A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone. Nat Protoc. 2010;5(3):550-560.
[10] Jia GQ, Zhang MM, Yang P, et al. Effects of the different culture and isolation methods on the growth, proliferation and biology characteristics of rat bone marrow mesenchymal stem cells. Sichuan Da Xue Xue Bao Yi Xue Ban. 2009;40(4): 719-723.
[11] 唐少锋,刘承杏.胎牛血清对胎鼠神经干细胞分化的影响[J].昆明医学院学报,2009,30(5):33-36.
[12] 刘媛,龙在云,曾琳,等.不同培养条件对神经干细胞分化的影响[J].中国临床康复,2003,7(31):4228-4229.
[13] Daniels DL, Eklof Spink K, Weis WI. Beta-catenin: molecular plasticity and drug design. Trends Biochem Sci. 2001;26(11): 672-678.
[14] Cavallo RA, Cox RT, Moline MM, et al. Drosophila Tcf and Groucho interact to repress Wingless signalling activity. Nature. 1998;395(6702):604-608.
[15] Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways. Science. 2004;303(5663):1483-1487.
[16] Wislet-Gendebien S, Hans G, Leprince P, et al. Plasticity of cultured mesenchymal stem cells: switch from nestin-positive to excitable neuron-like phenotype. Stem Cells. 2005;23(3): 392-402.
[17] 陈丹丹,付文玉,庄文欣,等.多种细胞因子诱导大鼠间充质干细胞向多巴胺能神经元分化[J].解剖学杂志,2011,34(5):584-587.
[18] Brunet N, Tarabal O, Portero-Otín M, et al. Survival and death of mature avian motoneurons in organotypic slice culture: trophic requirements for survival and different types of degeneration. J Comp Neurol. 2007;501(5):669-690.
[19] Li Q, Ping P, Jiang H, et al. Nerve conduit filled with GDNF gene-modified Schwann cells enhances regeneration of the peripheral nerve. Microsurgery. 2006;26(2):116-121.
[20] 吴晓娟,魏明发,柴成伟,等.大鼠骨髓间充质干细胞体外向肠神经细胞分化及胶质细胞源神经营养因子表达的变化[J].中华小儿外科杂志,2010,31(8):607-611.
[21] Tanaka J, Horiike Y, Matsuzaki M, et al. Protein synthesis and neurotrophin-dependent structural plasticity of single dendritic spines. Science. 2008;319(5870):1683-1687.
[22] Yoshii A, Constantine-Paton M. Postsynaptic BDNF-TrkB signaling in synapse maturation, plasticity, and disease. Dev Neurobiol. 2010;70(5):304-322.
[23] Song CW,Fang SQ,Lv G,et al.Gastrodin promotes the secretion of brain-derived neurotrophic factor in the injured spinal cord.Neural Regen Res. 2013;8 (15): 1383-1389.
[24] Chen BY, Wang X, Wang ZY, et al. Brain-derived neurotrophic factor stimulates proliferation and differentiation of neural stem cells, possibly by triggering the Wnt/β-catenin signaling pathway. J Neurosci Res. 2013;91(1):30-41.