Treatment Of Traumatic Brain Injury In Female Rats With Intravenous Administration Of Endothelial Progenitor Cells

PARTⅠ:The study of induction,differentiation and identification of endothelial progenitor cells from marrow stromal cellsObjective:To investigate the method for isolation and culture of bone marrow stromal cells(MSCs),furthermore these cells were suspended in medium supplemented with VEGF and bFGF for culturing,differentiating,proliferating and identification.Methods:Male rats aged 1 month were used to isolate MSCs from the femurs,The MSCs were cultured and purified by their characteristic of plastic adhesion in vitro.To characterize these cells,fluroscent cell sorting was performed using antibody against cell surface marker by flow cytometry and percentage of positive cells for CD90,CD31,CD34 were determined respectively.In order to obtain EPCs from MSCs,MSCs were treated with VEGF(20ng/ml) and bFGF(20ng/ml).After culture for five days,The cells were identified by flow cytometry using antibody against cell surface marker.To identify whether the cells express the gene of eNOS using the technique of PCR.Results:Assayed by flow cytometry,the third passage of cultured MSCs showed homogeneity as high as 95%.The percentage of positive cells for CD90,CD31,CD34 was 99%,3.4%,0.3%respectively.The percentage of cells that had the phynotype of endothelial progenitor cells induced by cytokines was identified.The percentage of positive cells for CD133, CD34,Flk-1 was 52%,33%,38%respectively.Using PCR technique showed the cells can express the gene of eNOS.Conclusion:High purifed MSCs can be obtained by adherent method.The cells induced from MSCs can express the phynotype of EPCs and have the function of EPCs. PARTⅡ:The study of endothelial progenitor cells home to traumatic brain injury tissue extraction in vitroObjectives:To investigate the capability of endothelial progenitor cells(EPCs) homing to traumatic brain injury tissue extraction(TBITE)in vitro.Methods:EPCs trial is experimental trial.MSCs and 3T3 trials are control trials.Placing these cells in the upper vessel of 24-well transvessle and placing 30%,20%,10%TBITE and common culture medium in the low vessel.The numbers of the cells in the low vessel after 30 minutes were calculated.Results:More cells were investigated in the trials of EPCs and MSCs at 30%,20%,10%TBITE.The capacity of homing between the two cells have no difference.EPCs have more powerful homing capacity at high TBITE concentrition.Conclusions:1.EPCs have more powerful homing capacity contrast to 3T3 cells for TBITE in vitro.2.EPCs and MSCs have similar homing capacity to TBITE.3.EPCs have more powerful homing capacity at high concentrition.4.There are much inflammatory factors in the TBITE,these factor contribute to EPCs' homing.PARTⅢ:Treatment of traumatic brain injury in female rats with intravenous administration of endothelial progenitor cells induced from marrow stromal cellsObjetive:To study the effect ofⅣ-injected endothelial progenitor cells induced from marrow stromal cells and the sites that the cells homed to.Methods:1.The model of fluid percussion injury:The injury site was on the left parietal cortex midway between the lambra and bregma,The impact pressure is 1.3～2.0atm.2.The groups of the animals:Female rats weighed 280～300g were randomly divided into four groups:1)Experimental trial: endothelial progenitor cells from male rats were injected into the tail vein of the female rats(n=6)24 hours after traumatic brain injury.2)Control trial 1:marrow stromal cells from male rats were injected into the tail vein of the female rats(n=6) 24 hours after traumatic brain injury.3)Control trial 2:3T3 cells were injected into the tail vein of the female rats(n=6)24 hours after traumatic brain injury.4)Control trial 3:endothelial progenitor cells were injected into the tail vein of the female rats after sham-injury.3.Cells transplantation:The cells number of EPCs,MSCs and 3T3 for every trial is 2×10~6.All the cells are the prelabeled by Brdu.4.mNSS evaluation:To evaluate the neurological function of the rats using modified neurologic severity score preinjury and at 2d,7d,14d,28d after transplanation.5.Histology evaluation:The distribution of male donor cells in brain,heart, lung,kidney,liver,spleen of the female recipient rats was measured by immunohistology chemical stain,that is single stain:Brdu and double stain: Brdu+BDNF;Brdu+Ⅷfactor.6.Fluorescent in site hybridyzation:Investigating the convinced distribution of the male donor cells using the specific Sry gene of male rat as a probe.Results:1.Brain injury model:There were no differences among groups which rats were injured.2.Evaluation of neurological function:Trials of experimental and control 1 significantly improved the rats functional outcomes contrast by trials of control 2(p＜0.05).But the outcome of the trials of experimental and control 1 had no difference.3.Histological and pathologic changes:There were traumatic tissue necrosis and gliocyte proliferation in all of the injured brain.4.Immunochemical stain:The transplanted cells in trials of experimental and control 1 successfully migrated into the injured brain and were preferentially localized around the injury site.The similar state were not seen in the trials of control 2 and control 3.More donor cells in the experimental trial join in angiogenesis than the trial of control 1.But expression of BDNF in the trials of the experimental and control 1 had no difference.The distibution at lung, liver,heart,kidney,spleen is 1.6%,0.3%,0.002%,0.01%and 0.5% respectively.5.Fluorescent in site hybridyzation:The donar cells were found more by the method of FISH than the mothod of immunochemical stain.The number of the latter is 30%of the first.Conclusions:1.Endothelial progenitor cells from MSCs can home to the site around the injury site.2.The cells can repair injured vein and enhance angiogenesis at injured site.3.EPCs have the same capacity of secreting the factor of BDNF as MSCs.4.The theory of treatment by EPCs:1)Enhancing blood supply to the injury site can save dying neurons.2)Secreting neural nutrition factors and improving the functions of neurons.3)Promoting neural stem cells in vivo to the injury site,proliferating and differentiation,reconstructing the new neural circle.4)VEGF can attract EPCs homing to the injury site and promote EPCs secreting VEGF and NO.NO can dilate artery to enhance the blood supply.