摘要
Abstract
BACKGROUND: It is of significance to study the direct differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into dopaminergic neurons and their functional characteristics for the treatment of neuropsychiatric disease such as Parkinson's disease with cell transplantation.OBJECTIVE: To induce the direct differentiation of hBMSCs into dopaminergic neurons by brain-derived neurotrophic factor (BDNF), forskolin, and dopamine, and to investigate the structural and functional characteristics of dopaminergic neurons. DESIGN: Randomized controlled observation.SETTING: Zhengzhou University and Henan University.MATERIALS: This study was performed in the laboratories of Zhengzhou University and Henan University from March 2005 to September 2006. Bone marrow was extracted from 15 healthy volunteers. All subjects provided the informed consent, and the experiment was approved by the local ethics committee. BDNF, dopamine, forskolin, and monoclonal antibody tyrosine hydroxylase were provided by Sigma Company, USA.METHODS: Mononuclear cells were obtained from human bone marrow by density gradient centrifugation, and BMSCs were purified with adherent culture. BMSCs were induced by BDNF (50_ μ mol/L), forskolin (10 μ mol/L), and dopamine (10 μ mol/L). After two weeks, electron microscope was adopted to observe cellular ultrastructure; immunocytochemical staining was adopted to observe the expression of NSE and TH proteins, and RT-PCR was adopted to detect expressions of Nurr1, Ptx3, Lmx1b, tyrosine hydroxylase mRNA during the differentiation into dopaminergic neurons. Moreover, non-induced cells were collected as the control group, and level of dopamine release was measured by high performance liquid chromatogram.MAIN OUTCOME MEASURES: Cellular ultramicrostructure; changes in the expression of NSE and TH protein; released level of dopamine.RESULTS: Cellular ultramicrostructure: After two weeks, a great quantity of concentrated cystiform-shaped rough endoplasmic reticulum, a lot of free ribosome, and some neurofilament appeared in cytoplasm. Changes in the expression of NSE and TH proteins: immunocytochemistry staining indicated the ratio of NSE-positive and TH-positive neural cells was significantly increased in different stages after induction (P<0.001). Changes of key transcription factors and tyrosine hydroxylase mRNA: RT-PCR indicated that all cells could express Nurr1, Ptx3, Lmx1b, and tyrosine hydroxylase mRNA. Level of dopamine release: After two weeks, level of dopamine release in the induced cells was significantly higher than that in the non-induced cells (P < 0.05).CONCLUSION: BDNF, forskolin, and dopamine can induce hBMSCs to differentiate into cells that have the structural and functional characteristics of dopaminergic neurons in vitro.关键词
多巴胺神经元/骨髓间充质干细胞/分化/脑源性神经营养因子/高效液相色谱分类
医药卫生
