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BACKGROUND: Glial cell line derived neurotrophic factor (GDNF) plays an important role in inducing the differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro and promoting the regeneration of neuron axons. OBJECTIVE: To observe BMSCs differentiation induced by over-expression of GDNF gene, detect synaptic function of cells and expression of Wnt signaling pathway components after differentiation, and preliminarily explore the mechanism of BMSCs differentiation into mature neurons. METHODS: Rat BMSCs were isolated and cultured, and further divided into recombinant adenovirus-containing GDNF gene transfection group (Ad-GDNFBMSCs), adenovirus transfection control group (Ad-BMSCs), and untransfected control group. The relative expression of GDNF gene in BMSCs of each group was detected by Q-PCR, and the expression of β-catenin, cyclin D1, NeuN and MAP-2 was detected by immunofluorescence technology in each group. High K+ stimulation induced cell depolarization response after differentiation, and FM4-64 marks synaptic vesicle activity of differentiated cells. RESULTS AND CONCLUSION: (1) The adenovirus-transfected gene had no significant negative effect on the proliferation of BMSCs. BMSCs could express endogenous GDNF gene continuously and at high levels. (2) Under the induction of GDNF gene, BMSCs could express neuron-specific protein NeuN after 3 days cultivation in vitro. The expression of β-catenin protein also could be detected in the cytoplasm of cells. After 7 days cultivation in vitro culture, BMSCs expressed the mature neuronal marker protein MAP-2, and the cell body contracted significantly. Neuron axon-like structures appeared around the cell body. Moreover, β-catenin and cyclin D1 were respectively detected in the cell cytoplasm and the nucleus, while the expression levels of NeuN, MAP-2, β-catenin, and cyclin D1 were not observed in Ad-BMSCs and untransfected control groups, and the cells remained spindle-shaped. (3) After 11 days of in vitro culture, the cells in the Ad-GDNF-BMSCs group showed typical neuronal processes or axons and connected to each other into a network structure, which could be labeled with FM4-64 to show red fluorescence, and induced by high K+ stimulation to induce action potentials in the cells. Synaptic vesicle activity in posterior axons showed FM4-64 red fluorescence gradually decaying. Under the same conditions, cells in the Ad-GDNF-BMSCs group and untransfected control group did not present FM4-64 fluorescently labeled synaptic vesicle activity. (4) Continuous GDNF induction can promote BMSCs differentiated into mature neurons with synaptic cycle function, and may be carried out through the classic Wnt/β-catenin signaling pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Olfactory ensheathing cell transplantation for treating spinal cord injury is an issue of concern, which mainly explores the changes of microenvironment after spinal cord injury. However, the effect of olfactory ensheathing cell transplantation on the ultrastructure of spinal cord after spinal cord injury is never reported. OBJECTIVE: To investigate the ultrastructure alterations of neurocytes, axons, myelin sheaths, synapses, and glial scar after spinal cord injury, and the effect of olfactory ensheathing cell transplantation on the protection and regeneration of nerve repair after spinal cord injury. METHODS: The study was approved by the Ethical Committee of Biomedicine of Medical Department of Xi'an Jiaotong University, approval No. 2018-2048. Twenty female Sprague-Dawley rats ware divided into three groups: blank control group (n=4, complete laminectomy of T10, partial laminectomy of T9and T11), DF12 group (n=8, cordotomy+injection of DF12 solution), and olfactory ensheathing cell transplantation group (n=8, cordotomy+olfactory ensheathing cell transplantation). The spinal cord was removed under anesthesia to observe the ultrastructure alterations of neurocytes under transmission electron microscope at 1, 7, 28 and 56 days after injury. RESULTS AND CONCLUSION: (1) Compared with the blank control group, the organelles in the neurons of the injured lesions were significantly reduced, and the obvious changes were found in the ultrastructure of axon, synapses and myelin sheath in the DF12 group. In the olfactory ensheathing cell transplantation group, the organelles in the neurons of the injured lesions were significantly increased with obvious nucleolus, the regeneration of axon, myelin sheath and synapses were significantly promoted, and the glial scar was significantly decreased. (2) The degree of reaction of the astrocytes and perlcytes in the olfactory ensheathing cell transplantation group was light. (3) These findings suggest that olfactory ensheathing cell transplantation can effectively protect the nerve tissues in the lesions after spinal cord injury, promote the regeneration of axon, myelin sheath and synapses, and inhibit the hyperplasia of astrocytes and pericytes, so that the post-injury microenvironment is available for the regeneration of neurons, axons and synapses. [ABSTRACT FROM AUTHOR]