| Backgroud and Objective:Cerebral vascular disease (CVD) is one of clinical common diseases,which ranks to the3rdleading cause of death to human being and the1stcause of death lead by all vascular diseases.70%of cerebral vaseulardiseases are ischemic and even up to85%in USA and Eeuropeancountries. Up to now, there is no better method to treat such patientsexcept thrombolytic therapy in the acute period. Over the last decade,cell-based therapy has been introduced as a promising attempt to improverecovery after ischemic stroke.Among the different cell types under investigation for stroke, bonemarrow mononuclear cells (BMMNCs) represent a particularly attractivetreatment option. BMMNCs can be easily obtained by bone marrowaspiration, do not require extensive preparation or cultivation, and permitautologous intravenous transplantation. BMMNCs are known to holdgreat potential as cell sources of transplantation therapy for ischemicstroke.The mechanism by which BMMNCs contribute to recovery afterstroke is still unclear, but current preclinical evidence indicates that themain mechanism of BMMNCs based therapy is not by direct cell replacement, but by trophic factors which modulate the post-ischemicinflammatory response, attenuate neuronal cell death and increaseangiogenesis finally improve neurological function. So the BMMNCswhich have greater capacity of trophic factors secretion could offerbetter effect in treating ischemic stroke.We first discovered in the previous research that bone marrowregenerative mononuclear cells (BMRMNCs) have greater capacity ofsecretion of trophic factors（such as bFGF、NGF、BDNF、VEGF）thanBMMNCs. These trophic factors have a strong protective effect oncerebral ischemic injury. So we speculate that BMRMNCs can migrate tothe cerebral ischemic aera then secrete trophic factors and modulate thepost-ischemic inflammatory response, attenuate neuronal cell death andincrease angiogenesis finally improve neurological function. In order toconfirm this speculation, we transplant BMRMNCs intravenously into thefocal cerebral ischemia model rats and observe the neurologicalfunctional improvement, secretion of trophic factors in the ischemic aera,and the impact of trophic factors to neuronal apoptosis, inflammatory andangiogenesis. This study will confirm that due to the greater capacity ofsecretion of trophic factors, BMRMNCs offer better effect in treatingischemic stroke, BMRMNCs could be regarded as the most idealpromising multipotent candidat for the treatment of ischemic stroke. Matetials&Methods:Establish rat bone marrow depression and regeneration model andthen obtain the BMRMNCs；Inorder to achive reliable and effctivemiddle cerebral artery occlusion (MCAO) and reperfusion model wemodify the method and confirm the result; We transplant intravenouslythe BMRMNCs into MCAO model, then survey their survivorship andeffect in promoting brain repair and functional recovery，in further step toinvestigate the value and the mechnism of BMRMNCs in MCAOtreatment. The study inecudes five parts:1. Rat bone marrow depression and regeneration model wasestablished by injection of150mg/kg5-fluorouracil (5-Fu) via tailvein．Blood samples were taken before5-Fu injection and3d,7d，11dand14d after5-Fu injection，bone marrow samples were obtained aftersacrifice of rat．The changes of blood samples and bone marrowsamples during bone marrow depression and regeneration weredynamically observed．2. We use the shuttled tip suture-embolus to make the model ofMCAO reperfusion and evaluate this modified method by success rateand stabilization.3. In order to understand the biology function of BMRMNCs, weanalyze BMRMNCs’ seereting level of trophic factors in vitro and flowcytometric analysis was performed to evaluate the surface markers of BMRMNCs.4. The mechanisms regulating BMRMNCs migration andaccumulation in the injured brain remain to be revealed. we track theBMRMNCs after transplantaion and try to understand the mechanismswhy BMRMNCs can survive, migrate toward injured tissue, andpromote recovery of neurological function following cerebral infarction.5. we transplanted BMRMNCs intravenously into the focal cerebralischemia model rats and observe the neurological functional improvement,secretion of trophic factors in the ischemic aera, and the impact of trophicfactors to neuronal apoptosis, inflammatory and angiogenesis. This studywill try to understand the mechanism that by greater capacity of trophicfactors secretion BMMNCs contribute to recovery after stroke.Results:1. The numbers of erythrocytes， mononuclear cells andmegakaryocytes dramatically decreased in model after injection of5-Fu,in3d，and to a nadir in7d and restore to the normal level in14d, Thesephenomena indicated that the rat bone marrow depression andregeneration model was successful.2. The modified method of MCAO is proved to be stable andrepeatable, and cerevral infraction consistent with the clinicalpathological process. It is perfect model to study focal cerebral ischemic injury.3. BMRMNCs can secret more trophic factors（such as bFGF、NGF、BDNF、VEGF） in vitro, and the rate of CD34、CD45、CD90postive cells increase which indicates that the content of stem cellsincrease.4. After transplantaion, more BMRMNCs can migrate towardischemic tissue and survive. The enhancement of migration capacity ispossible related with the improvement of expression of CXCR4.5. There was significant neurological function improvement in ratstreated with BMRMNCs compared with BMMNCs. After transplantationof BMRMNCs we found that the level of trophic factors and number ofcerebral microvessel in penumbral regions increased; level ofinflammatory factors and numbers of apoptotic cells decreased; rate oftransdifferentiation to neuron and neuroglial cell was very low. Thesephenomena indicated that it is trophic factors that modulate thepost-ischemic inflammatory response, attenuate neuronal cell death andincrease angiogenesis finally improve neurological function.Conclusion:Compared to BMMNCs, BMRMNCs can secret more trophic factorssuch as (bFGF、NGF、BDNF、VEGF） and have gerater migration capacity in vitro. In rats subjected to MCAO, intravenously injectedBMRMNCs can pass through blood brain barrier, migrate selectively,target damaged brain and survive. BMRMNCs can reduce neuronalapoptosis and upregulate expression level of trophic factors inpenumbral regions, enhances angiogenesis, decrease the infarct volumesand brain edema, and promote the neurological functional improvement,BMRMNCs have a better therapeutic efficacy than BMMNCs althoughthey both have effect for cerebral infarction therapy. BMRMNCs could beregarded as the most ideal promising multipotent candidat for thetreatment of ischemic stroke... |
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