| Objective:To explore the efficacy and safety of intravenous bone marrow-derived mesenchymal stem cells (BMSCs) for the treatment of ischemia-reperfusion injury in rats and investigate its possible mechanism.Methods:1. Take the long bone marrow of SD rats as a source of donor, and BMSCs were isolated using density gradient centrifugation combined with adherent screening method. We have identified the cells by the cell morphology and the surface markers.10mg/L BrdU was used for labeling before BMSCs transplantation.2. SD rats were used to establish the middle cerebral artery ischemia-reperfusion model with modified suture method,and then the MCAO model was identified by TTC staining.3. The models were randomly divided into three groups.①Group A:7days after injury, rats in group A were given3×10*6BMSCs through the vena caudalis.②Group B:7days after injury, rats in group A were given1×10*6BMSCs through the vena caudalis.③Group C:Rats in the model group were left intact.4.①The ability of moving were evaluated with modified Neurological severity scores.②The ability of learning and memory were evaluated with the Morris water maze.5. Rats were executed in7,14,30d after transplantation to the immunohistochemical staining including CD11b/MPO-positive cells,BrdU single-staining,BDNF single-staining, Brdu+MAP-2,Brdu+GFAP,Brdu+vWF,Brdu+VEGF double-staining. BMSCs for transplantation were observed the survival and differentiation in host brain by Immunohistochemical staining.Results:1. BMSCs were isolated using density gradient centrifugation combined with adherent screening method.The3rd generation cells was detected by flow cytometer.The expression of CD90, CD71were99.74%.98.93%. The expression of CD45、CD34、CD14were0.36%、0.32%、0.45%. It’s shown that these cells were most in undifferentiated state,and having the attached characteristics to the plastic substrate.It’s in line with the characteristics of BMSCs, confirmed the cells we separated were BMSCs;2.The rats developed typical symptoms of neurological impairment afer they were subjected to MCAO. TTC staining shows ischemic regions were pale,which proved the model is success.3.Aafter transplantation of the BMSCs,group A’s recovery of the neurological deficit is more apparently than group C (P＜0.01); the recovery of rats in group A was more apparently than than group B (P＜0.01); and had no significant difference between group B and group C (P>0.05);4.Double-staining cells of immunohistochemistry could be found at the center of damage site, choroid plexus, around ependymal layer and the perivascular. BrdU+cells and Immunohistochemical double staining positive cells(Brdu+MAP-2, Brdu+GFAP, Brdu+vWF, Brdu+VEGF) were seen in group A and group B.Conclusion:1. BMSCs could separated and amplification in vitro.2. Improved suture method is simple and reliable.It can be used in the research of focal cerebral ischemia-reperfusion injury.3.BMSCs transplanted through vena caudalis can survive and differentiate in the host brain damage region. BMSCs transplantation is able to improve the recovery of the neural function of cerebral ischemia injured rats. The mechanism may be related to the transplanted cells differentiating into neuron-like and glial-like cells, angiogenesis, and secretion or promoting the secretion of neurotrophic factors of host cells.4. The efficacy is related to the number of BMSCs,3×10*6has a better therapeutic effect.5. Immunological rejection and tumour formation do not detected in host. |
PDF Full Text Request