| Objective:On the basis of taking advantage of bone marrow-derived mesenchymal stem cells (BMSCs) for the treatment applying spinal cord injury rat model against spinal cord injury device model, it will be under close observation of the spinal cord injury in the protection and repair, and through its migrate to the migration of spinal cord injury, apoptosis inhibitory neurons to investigate the mechanism of injury repair.Research:Methods:Isolating and purifying the BMSCs according to the adherent traits of MSCs in vitro. Through morphological changes and the cell cycle determined by flow cytometry, MTT assay of its growth characteristics and flow cytometry detection of cell surface markers identified by the separation of BMSCs; Made against the use of equipment, application of an improved animal model of Allen prepared large mouse model of spinal cord injury; On the basis of the improved Tarlov in accordance with the evaluation criteria, observing MSCs on spinal cord injury model in rats. The BMSCs were observed on the untreated after treatment and pathological changes in rats. Using confocal laser scanning microscope to observe whether BMSCs labeled by vitro DAPI which was injected into the tail vein after the injury convert and settle in; Immunohistochemical method is used to observe the BMSCs after treatment of Bcl-2, Bax and caspase-3 expression. The spinal cords are maked into frozen section, which are examined under the laser scanning confocal microscope for the condition that the transplanted BMSCs express the neuron-specific protein-NSE.Results:1.The isolation and purification of BMSCs and their morphological characteristics in vitro part. After being cultured, the cell morphology became more consistent and in which the majority is the slender spindle cells; It is evidenced by the MTT method of mapping the growth of curves that bone marrow stromal cell line is very strong in vitro proliferative activity; the flow cytometry detects that 85.87%of the cells are in G0-G1 phase, which shows that the detected BMSCs were characterised the growth of stem cells. There is no exact expression with the three generations of rat BMSCs in vitro part of the basic non-CD34, CD45, and CD44.2. Effects on BMSCs for the treatment of spinal cord injury in rats. In accordance with the modified Tarlov criteria function,15 days after surgery, the function scores compared with the model group's scores was significantly increased; 7,15,30 days after the surgery, treatment group's biopsy compared with the model groups'were significant recovery.3. BMSCs in rats with spinal cord injury-rich region It is found by confocal laser scanning microscopy that with injection of DAPI labeled BMSCs,5 days'vascular injury in the spinal cord it comes the fluorescent markers of BMSCs; The 10 days, after injection, around vascular injury and its surrounding tissue dispersion of BMSCs will be observed; On the 15 days after injury, there are fluorescent-labeled BMSCs wide disperse; Normal rats group which are injected for 5 days,10 days,15 days, wont be found with the DAPI labeled BMSCs.4. The inhibiting effect of BMSCs on neuronal cell apoptosis.7 days after the surgery, treatment group 2s'(BMSCs local transplantation group,) bax, caspase-3 positive cells less than the control group 2s' (surgery alone group) (P<0.05), treatment groupl's (BMSCs vein graft group) Bcl-2 positive cells more than control group2s'(P<0.05); 15 days after transplantation, treatment group 1 and treatment group 2, the expression of bax, caspase-3 positive cells less than control group2s' (P<0.05), treatment group 1 and treatment groups 2s'Bcl-2 positive cells more than control group 2s'(P<0.05); 30 days after transplantation, the expression of bax-positive cells in treatment group 1 less than which in control group 2 (P<0.05), Bcl-2 positive cells of treatment group 1> treatmentgroup 2 more than control group 2s'(P<0.05).5. The transplanted BMSCs express the neuron-specific protein-NSE: 15 days after transplantation, under the laser scanning confocal microscope, were observed BrdU/NSE double labeling cells.Conclusion:The success of this experiment lays in Purification and amplification in vitro of BMSCs; It managed to maintain its original characteristics and differentiation patterns after passaged repeatedly;With BMSCs tail vein injected into the local site of injury after spinal cord injury it can promote the recovery of motor function in rats, and repair large rats with spinal cord injury; By BMSCs tail vein injected into the local site of injury, the transplanted BMSCs into the anchor. It can be made to the damage and settle in tissue chemokine after BMSCs and damage parts of the tail vein after local were injcted; the vivo experiments show that BMSCs can inhibit caspase-3, Bax expression and increases Bcl-2 expression to promote the repair of spinal cord injury. This study not only enriches the theory of BMSCs in tissue repair and experimental data, but also provide data BMSCs used in clinical and theoretical basis.
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