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Rat bone marrow mesenchymal stem cell culture, purification and identification [Objective] investigate SD rat bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Stem Cells MSCs) were isolated, purified and passaged, identification, marking method to observe the biology of MSCs characteristics for further treatment using MSCs transplantation in rats with vascular dementia (Vascular dementia, VD) model basis. [Methods] SD rats were sacrificed by cervical dislocation, the femur bone marrow was collected using whole bone marrow adherent culture purified MSCs. By observing the primary and passaged MSCs after cell morphology, growth, growth curve plotted MSCs, MSCs understand the growth characteristics, morphology and proliferation. MSCs was measured by flow cytometry surface markers and in vitro induce MSCs to differentiate into osteoblasts on MSCs were identified. [Results] isolated and cultured MSCs fusiform morphology under the microscope for the growth of adherent fibroblast-like cells, the first passaged MSCs cells tend to form homogeneous, showing swirl-like or daisy-like growth. After passage MSCs grow rapidly the first 1-6 days, 7-8 days, because of the increase in cell density and thus the phenomenon of contact inhibition appears to slow down growth. With the proliferation of MSCs passaged increase in the number gradually decreased. Flow cytometry was the third generation of rat bone marrow MSCs found almost uniformly express CD 44, CD90, do not express CD34. Vitro osteogenic MSCs can differentiate into osteoblasts, Alizarin red S staining positive calcium nodules, alkaline phosphatase staining (alkaline phosphatase, ALP) show elevated alkaline phosphatase activity. [Conclusion] combined with whole bone marrow adherent cells were isolated, cultured and passaged MSCs SD rats with high purity, vigor strong, stable traits. Vascular dementia rat model of preparation [Objective] To explore the ideal preparation method VD animal model for the next production VD MSCs transplantation animal model. [Method] Firstly, the Morris water maze in SD rats were screened to remove 95% of the reference value is not within the scope of SD rats after screening out 79 rats were randomly divided into two groups: sham operation group 10, model 69 only. Model group using a modified two vascular ligation (2-VO) - interval of three days of bilateral carotid artery ligation were prepared by the method VD VD model rats, sham-operated group subjected to the same surgical procedure, but not bilateral carotid ligation artery, caged 4 weeks (w) after the determination by Morris water maze test of spatial learning and memory in rats two changes. [Results] 84 rats by water maze screening to remove four, died 1. Will be selected for the experiment 79 rats were randomly divided into sham operation group 10, model 69. Before modeling sham group and model group, the escape latency in the residence time and the platform quadrant no difference, p value greater than 0.05. After modeling the sham group died an infection, improper feeding killed two, I 7. Model rats killed 33, survived 36, excluding a limb movement disorder, three less than VD standards, the present study were successfully produced a 32 SD rats VD model. After modeling the escape latency model group was significantly longer than the sham group, the platform quadrant residence time was significantly shorter than the sham group, p value less than 0.01 (Table 2). The experimental model of SD rats produced survival 52.17%, 91.43% rate into a mold. [Conclusion] The 2-VO improved the VD model has a lower mortality rate, a higher rate into a mold, easy operation, good repeatability, rat VD model is an ideal method. Mannitol on bone marrow mesenchymal stem cell transplantation in the treatment of vascular disease rat model of efficacy stay [Objective] mannitol preliminary study on bone marrow mesenchymal stem cell transplantation for treatment of VD rats therapeutic effect. [Methods] After the modeling medium VD rats were randomly divided into eight groups (injection 1ml serum-free medium), MSCs group 11 (injecting 1 × 106 个 MSCs1ml) and mannitol MSCs transplantation in the treatment group 9 ( 1.5g/kg mannitol injection after injection in 10-30 minutes 1 × 106 个 MSCs1ml), caged 4 weeks after transplantation by Morris water maze test for detection of these three groups and the sham-operated rats behavioral changes , each group was detected using the rat model ElISA frontal cortex and cerebrospinal fluid levels of VEGF, HE staining pathological structural changes in the frontal cortex. [Results] MSCs after intravenous transplantation 4W, behavioral results mannitol MSCs group and MSCs group escape latency was significantly shorter than the medium, platform quadrant mean residence time extension (p lt; 0.02), mannitol MSCs MSCs transplantation group than in the group More significantly shorter platform quadrant mean residence time longer (p lt; 0.03); mannitol MSCs group, MSCs group frontal cortex, CSF VEGF levels in the medium group of high-average ratio (p lt; 0.001); in the frontal cortex , mannitol MSCs group, MSCs group mean VEGF levels in the medium group than high VEGF levels mannitol MSCs group average ratio MSCs group, the difference was statistically significant, p are 0.000; in the cerebrospinal fluid, mannitol MSCs group were compared with MSCs group, medium group VEGF levels are higher (p = 0.000), MSCs group than in the medium group, the mean high levels (p = 0.021). Mannitol MSCs group and MSCs group frontal cortex pathology film showed the pathological injury group compared with medium light, larger number of neurons, neuronal swelling, depigmentation and nuclear condensation phenomenon is reduced, while mannitol MSCs group improved more obvious. Conclusion MSCs intravenously make VD rat model to improve spatial memory and learning ability; mannitol pretreatment after intravenous injection of MSCs group VD rats improved spatial memory and learning ability MSCs group than alone more obvious. Possible mechanisms with mannitol and joint use of MSCs after intravenous injection increased intracranial increased VEGF expression and neuronal cell protective effects.
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