| Objecitive:①To establish the method of the isolation, cultivation and gene transfection of bone mesenchymal stem cells (BMSCs).②To evaluate the therapeutic effect of BMSCs, Noggin and brain-derived neurotrophic factor (BDNF) modified BMSCs, hydroxysafflor yellow A (HSYA) and repetitive transcranial magnetic stimulation (rTMS) on the vascular dementia (VaD) rats by the Morris water maze and the vascular endothelial growth factor (VEGF) expression in the hippocampus. To analyze the physiological and molecular mechanism of the learning and memory improvement, indicated by the long-term potentiation (LTP), the BDNF and different N-methyl-D-aspartic acid receptor (NMDAR) subunits expression in the hippocampus.Method:①BMSCs were isolated from SD rats bone marrow and purified through adherence ability. These cells were identified as BMSCs by their phenotypical properties and their ability to differentiate into osteocyte and adipocyte.②Ad-GFP-Noggin gene and Ad-GFP-BDNF gene were transfected to the BMSCs. The target proteins were measured with Western blotting.③The rats were randomly divided into ten groups, including normal control, sham operation, VaD model, PBS, NS, BMSCs, Noggin modified BMSCs, BDNF modified BMSCs, HSYA and rTMS. The VaD model rats were made by two vessel occlusion (2VO) method.④BMSCs, Noggin modified BMSCs, BDNF modified BMSCs, HSYA(2weeks duration) and rTMS(4weeks duration) were administered in one week after the2VO.⑤The spatial learning and memory of all groups were measured by Morris water maze in five weeks after the operation. Then the CA3-to-CA1LTP were measured. The VEGF, BDNF, NR1, NR2A and NR2B expression in the hippocampus were measured with Western blotting. The pathologic changes were observed by hematoxylin and eosin stain. The VEGF and NR1expression in the hippocampus were also observed by immunohistochemistry.Results:①The method that isolation and cultivation of BMSCs through the adherence ability could effectively purify the BMSCs, which identified by the morphology, flow cytometry, osteogenic differentiation and adipogenic differentiation. ②The BMSCs modified by Ad-GFP-Noggin and Ad-GFP-BDNF could express Noggin and BDNF respectively with high efficiency.③The2VO model could mimic the pathological features of vascular dementia in human, in which could be improved by BMSCs transplantation (modified with or without Noggin and BDNF), HS YA and rTMS.④All therapeutic groups could improve the spatial learning and memory of rats in the Morris Water Maze tests, and the improvement was greater in Noggin and BDNF modified BMSCs groups than that in BMSCs groups.⑤The VEGF expression in the hippocampus was slightly higher after2VO. HSYA and BDNF modified BMSCs transplantation could significantly up-regulate the VEGF expression in the hippocampus.⑥All the therapeutic groups could enhance the CA3-to-CA1LTP, especially the Noggin or BDNF modified BMSCs transplantation and the rTMS groups.⑦All the therapeutic groups could increase the BDNF, NR1and NR2B expression, but not NR2A, in the hippocampus, especially the BDNF modified BMSCs transplantation and the rTMS groups.Conclusion:①BMSCs could be isolated and purified from rat bone marrow through the adherence ability and subculture.②Both Noggin and BDNF play a crucial role on the survival and differatiation of the BMSCs. The method of neurotrophic factors gene transfected BMSCs is a very hopeful therapeutic strategy in the nervous system diseases.③The BDNF modified BMSCs transplantation was proved to be better in improving the memory and synaptic efficacy, and promote the angiogenesis, compared with the BMSCs transplantation or Noggin modified BMSCs transplantation. Therefore the BDNF modified BMSCs transplantation is a promising therapeutic method in the treatment of VaD.④HSYA could promote the angiogenesis in the hippocampus, and rTMS could significantly enhance the synaptic plasticity, so combination HSYA and rTMS is worthwhile of applying in the treatment of VaD patients.⑤The formation of LTP is associated with BDNF, NR1and NR2B, but not NR2A, in the hippocampus. BMSCs (modified with or without Noggin and BDNF) transplantation, HSYA and rTMS improved the spatial learning and memory of the VaD rats through the BDNF-NMDAR (NR1/NR2B) dependent LTP. |
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