Intravenous Administration Of Marrow Stromal Cells Increase The Expression Of Vascular Endothelial Growth Factor After Cerebral Infarction In Rats

ObjectiveMarrow stromal cells ( MSCs) are non - haematogenesis stem cells that have multipotential. Researchs found that the injected marrow stromal cells can migrate into ischemic brain and differentiate into neuron - like cells or glial cells, and marrow stromal cells may secrete sorts of cytokine, which have important role in the brain injury reparative process. As vascular endothelial cell mito-gen, Vascular endothelial growth factor (VEGF) may promote angiogenesis and neoangiogenic. Researchs reported that the expression of vascular endothelial growth factor were enhanced after brain ischemia in rats, which affected patient prognosis after cerebral infarction. Our objective were to observe the distribution of the administrated cells in rats brain and the expression of vascular endothelial growth factor in rats brain after intravenous administration of marrow stromal cells for middle cerebral artery occlusion reperfusion model in rats, and explore the mechanism of marrow stromal cells theraphy for cerebral infarction.MethodTo establish 45 rats for middle cerebral artery occlusion (MCAO) reperfusion model according to Longa reporting: middle cerebral artery occlusion was attained with a 0.26mm diameter nylon suture, after 2 hours of middle cerebralartery occlusion, reperfusion was performed by withdrawl of the suture. Animal models were divived sandomly into three groups; groups treated with MSCs, control groups treated with saline, control groups treated with alone MCAO, every groups were divided into three time points: day 3, day 7, day 14. To demonstrate the form of brain infarction focal, the removal brain were stained immediately by tetrazolium chloride after middle cerebral artery occlusion, active tissue were stained red, infarction tissue were not stained.As described by Friedenstein, marrow stromal cells were isolated and obtained from two months Wistar rat (weight 250 g) postlimb bone marrow, filtered and centrifuged, cells were plated at a concentration of 2 x 10 cells/cm 25 cm2 tissue culture flasks in Dulbecco modified Eagle medium and were supplemented with 20% fetal bovine serum, 100 units/ml penicillin, 100|xg/ml streptomycin, and cultured in 371 ,5%CO2, saturated humidity incubator. The cells were observed under inverted phase contrast microscope. Primary cells were digested and passaged on day 8 ~ 10 (80% confluence). Subculture cells were cultured continuely. CD34,CD44,CD54 immuncytochemical staining were completed to identify marrow stromal cells.3 passage to 5 passage MSCs were prelabeled 24 hours before intravenous administration by supplementing bromodeoxyuridine ( BrdU, a thymidine analog that labels newly synthesized DNA) (3jxg/ml) into medium. Every rats received 3 x 106 prelabeled MSCs by tail vein ; Using immunohistochemical staining to observe the distribution of BrdU - immunreactive cells in brain and the expression of vascular endothelial growth factor in rats brain.ResultMarrow stromal cells cultured in vitro were polymorphic and could adhere the wall. MSCs could expand rapidly. The biological properties of subculture cells did not significantly change after passages. The result of immuncytochemical staining were CD34( - ) ,%CD44( + ) ,CD54( + ).BrdU - immunreactive cells were distributed mainly surrounding infarction area in the brain sections treated with MSCs.