Transplantation of bone marrow mesenchymal stem cells overexpressing glial cell line derived neurotrophic factor gene for spinal cord injury / 中国组织工程研究

ABSTRACT

BACKGROUND: Glial cell line derived neurotrophic factor (GDNF) plays an important role in inducing differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro and promoting neurological function recovery in rats with spinal cord injury. OBJECTIVE: To observe potential molecular mechanisms of differentiation of BMSCs overexpressing GDNF gene and promoting neurological function recovery after spinal cord injury in rats. METHODS: (1) BMSCs transfected with recombinant target gene adenovirus were divided into Ad-GDNF-GFP transfection group, Ad-GFP transfection group and non-transfection group. Microtubule-associated protein 2 and neuron-specific enolase expression levels were detected by immunofluorescence in each group. Western blot assay was used to detect the expression of GDNF, Wnt3a and Wnt7a protein in each group. (2) The rat spinal cord injury model was prepared by modified Allen method. The 45 Sprague-Dawley rat models were randomly divided into three groups. GDNF-BMSCs, BMSCs and PBS were transplanted into the site of spinal cord injury. The motor function recovery of rats was evaluated 4 weeks after operation. The morphological changes of spinal cord were observed by hematoxylin-eosin staining. The local neuron-specific enolase, Synapsin I and glial fibrillary acidic protein were analyzed with immunohistochemistry. The expression levels of Bcl-2 and tumor necrosis factor-α protein were detected by western blot assay. RESULTS AND CONCLUSION: (1) BMSCs overexpressing GDNF gene could differentiate into neuron-like cells and express neuron-specific enolase and microtubule-associated protein 2 in vitro in the Ad-GDNF-GFP transfection group. The expression of Wnt3a and Wnt7a protein was significantly higher in the Ad-GDNF-GFP transfection group than in the Ad-GFP transfection group and non-transfection group. (2) The Basso, Beattie and Bresnahan score in GDNF-BMSCs group was significantly increased and the stenosis area was significantly reduced at 4 weeks after transplantation. The expression of glial fibrillary acidic protein and tumor necrosis factor-α in GDNF-BMSCs group was significantly lower than that in BMSCs and PBS transplantation groups, but the expression levels of neuron-specific enolase, Synapsin I and Bcl-2 were significantly higher than those in the BMSCs and PBS transplantation groups. (3) Wnt signaling pathway participates in the procession of differentiating into mature neurons derived from BMSCs overexpressing GDNF gene. After transplantation, the effects of BMSCs transplantation on spinal cord injury were improved by decreasing local inflammatory reaction, apoptosis and glial scar formation and promoting axonal regeneration.