The Use Of Mannitol For Bone Marrow Mesenchymal Stem Cells Transplantation

PARTⅠthe Isolation,Cultivation and Identification of Rat Bone Marrow-derived Mesenchymal Stem Cells【Objective】To explore the separation, purification, propagation and identification of rat mesenchymal stem mesenchymal stem cells (Bone marrow, BMSCs) in vitro, to provide a stable function for BMSCs transplantation .【Method】Separate the both femurs of infantile SD rat, separate and purify BMSCs with the method combinated whole bone marrow cultivation and adherent cell in aseptic condition. When 80%-90% of the plastic culture bottom was covered, digest the cells with 0.25% trypsin digestion 30-60s and passage the cells.Use the inverted phase contrast microscope to observate the morphology of primary cells and passaged cells. The amplificated BMSCs were identify by osteogenic differentiation and Flow Cytometer.【Results】Rat BMSCs can be isolated , cultured and amplified in vitro. They growed as adherence cells,and exhibited a spindle-shaped like fibroblast.spindle shape.Their morphology become similar gradually with passagions. Alizarin red staining showed the calcium mineralization positive.The test of alkaline phosphatase activities showed positive result. Use the Flow Cytometer detective surface markers of BMSCs .Almost all cells express CD44 and CD90.The positive rates were 89.4% and 99.2% ,while the positive rate of CD34 was 1.82% only.【Conclusion】The method combinated whole bone marrow cultivation and adherent cell can successfully construct a system about rat BMSCs isolation and cultivation in vitro, besides the cultured BMSCs were pure, activity and steady. BMSCs can be induced into osteoblasts because of the multiple differentiation potential.PARTⅡPreparation of Vascular Dementia Model in Rats【Objective】To explore the method for an animal model of vascular dementia (VD) and prepare the certain animal model of VD for next step.【Methods】The models of VD rats were established by permanent ligation of bilateral common carotid arteries at an interval of 3 days for each artery. Cognition of rats were tested by the Morris water maze.【Results】Survival rate of VD rates is 52.17% in 4 weeks .Compared with sham group, the created VD rat model group showed apparent cognitive dysfunction.【Conclusions】This improved method for model of VD is reliable, reproducible, and stable. It has the practicability and reliability and can be used in basic experiment study about VD.PARTⅢObservation on the Curative Effect of MannitolPretreatment and Transplantation of Bone Marrow-derived Mesenchymal Stem Cells on the Vascular Dementia Rats【Objective】To study the influence of mannitol pretreatmenton on cognition and the expression of brain-derived neurotrophic factor(BDNF) of vascular dementia (VD) rats after the vein transplantation of bone marrow mesenchymal stem cells(BMSCs) .【Methods】BMSCs(1×106/mL,1ml) were injected into caudal vein of Vascular dementia rat in 10-30mins after mannitol pretreatmenton (1.5g.kg-1weight).After 4 weeks, the cognitions of VD rats were tested by the Morris water maze,and the expression of BDNF of brain was detected by enzyme-linked immunosorbent assay .the cognition of VD rat were observe and change of BDNF in brain were detected by water maze and enzyme-linked immunosorbent assay .【Results】Morris water maze : Comparing to culture media group, the escape latency decreased significantly(P<0. 05) and platform quadrant residence time increased significantly(P<0. 05) in BMSCs transfer group and mannitol + BMSCs transfer group. Comparing to BMSCs transfer group, the escape latency decreased significantly(P<0. 01) and platform quadrant residence time increased significantly(P<0. 05) in mannitol + BMSCs transfer group. The expression of BDNF :Comparing to culture media group,the expression of BDNF(P<0.01) increased significantly(P<0. 01) in BMSCs transfer group and mannitol + BMSCs transfer group. Comparing to BMSCs transfer group,the expression of BDNF increased significantly(P<0. 01) in mannitol + BMSCs transfer group.【Conclusion】The combination of mannitol and BMSCs transplantation significantly improved cognition and the expression of BDNF in brain tissue in vascular dementia rats.