| Objectives:(1) To isolate, culture and identify the BMSCs from the SD rats.To induce and differentiate them into Nestin-positive BMSCs. And tansfected the Ang-1 into the cells by molecular biology. (2)To establish the models of clip compressive thoracic 10 spinal cord injury in the SD rats and investigate the feasibility of the models. (3) To look forward to the possibility of the nestin-positive BMSCs differentiate into neuron in spinal cord injury, and to look forward to the protection of Ang-1 about the nestin-positive BMSCs, the neogenetic and residual neuron in spinal cord injury by tansplanted the nestin-positive BMSCs modified by Ang-1 gene.Methods:(1)We isolated and cultured the BMSCs by whole bone marrow adherence method from tibias and femurs in SD rats,We observed the morphology of BMSCs by inverted microscope. We performed flow cytometric analysis, using a large monoclonal antibody panel:CD45, CD34, CD29, CD90,and we induced the BMSCs into osteoblast cells,adipocyte and neuron-like cells.In the end we induced the BMSCs into the other cells that the surface marker were positive of Nestin.Then we tansfected the Ang-1 gene into the cells by adenovirus vector, and detected the cells expressed Ang-1 through Weston blot technology. (2)We established graded model of clip compressive thoracic 10 spinal cord injury in the SD rats. One week later, we used the methods based on the assessment of the motor function, neurophysiology monitoring,and pathology test to investigate the feasibility of the model.(3)Rats of randomized grouping were successfully local injected by the certain suspensions a week post-operation, then we assessed the correlated function of Ang-1 and Nestin positive BMSCs to the spine by the methods based on the assessment of the motor function, neurophysiology monitoring, and pathology test after one to four weeks. Results:(1) BMSCs which was isolated and cultured by the whole bone marrow culture and adherence method, inereased lastingly and had a fast multiplication speed in vitro.The purity quotient of BMSCs after the third generation. BMSCs phenotype was considered positive for CD29, CD90 after flow cytometric analysis, and negative for the other monoclonals.The positive rate of BMSCs phenotype was out of 99%. They could be differentiated into osteoblast cells, adipocyte and neuron-like cells in vitro. The result of weston blot manifest that the Nestin-positive BMSCs transfected by Ang-1 can secreted the angiopoitin protein. (2) Most of the rats became paraplegia after spinal cord injury(BBB<4), the wave shape of CSEP could not be evoked, a lot of inflammatory cells infiltrated the spinal cord which was cytoplasmic vacuolation.(3) Certain neurologial recovery occur in group B, C at 1,2,4 weeks after transplantation, but group A was not. BBB showed significant difference between group B and group C (p<0.05), The CSEP also showed significant difference between group B and group C (p<0.05). After transplanting in group B and C,the cells labled BrdU, Nestin, NSE could be observed in the injury spine,and the cells labled BrdU could be observed around blood vessel. The number of cells labled BrdU, Nestin, NSE reduced gradually in 4 weeks, but group C remain a certain amount of positive cells,and group C showed significant more than group B (p<0.05).Conclusion:(1)We could harvested the BMSCs which was isolated and cultured by the whole bone marrow culture and adherence method.It was multipotentiality and could be differentiated the BMSCs into Nestin positive cells. we could successfully tansfected the Ang-1 into the nestin-positive BMSCs by molecular biology. (2)The models of SD rats spinal cord injury was successfully established. (3)The Nestin-positive BMSCs tranfected by Ang-1 had the ability to differentiate into neuron, and they restituted and protected the Nestin-positive BMSCs and the nerve cells, cured the injury spine.
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