| Background: In the therapeutic studies on the treatment of ischemic cerebrovasculardiseases, the neuroprotective theory applied into basic researches and animal experiments hasachieved satisfactory results, but has no obvious efficacy in clinical trials. Although thethrombolytic therapy can be effective to restore the regional cerebral blood flow and save theischemic penumbra tissue to promote the recovery of neurological function, it has beenlimited in the widely clinical application owing to the short time window (≤4.5h). Therefore,researchers begin to extend to the protection strategy of the neurovascular unit from thesimple protection of nerves or blood vessels. ABPN proposed an application for establishingvascular neurology and got an approval in2003. After that, the protection strategy of theneurovascular unit is to solve the plight of the treatment of cerebral ischemia.In the neurovascular unit protection strategy, researchers begin to treat neurons, glialcells, microvascular endothelial cells and matrix as a whole from the past concept which waspaid too much attention to the protection of neurons or blood vessels. As one of the ways tostudy this strategy, EPCs transplantation has achieved some results in the treatment ofischemic cerebrovascular diseases. EPCs transplantation can play protective roles in repairingthe injured vascular endothel, promoting angiogenesis and vasculogenesis in the treatment ofischemic cerebrovascular diseases, which has been confirmed in the animal experiments. Atthe same time, some researchers speculate that EPCs transplantation may also play aneuroprotective role by the paracrine of cytokines (VEGF and BDNF HGF, etc.), and this roleis independent of angiogenesis and vasculogenesis.So we hypothesize that EPCstransplantation may play a protective role in the neurovascular unit, and one of themechanisms of this protective effect is mediated by the paracrine of VEGF.VEGF is known as vascular endothelial growth factor,which promotes endothelial cellproliferation and angiogenesis by binding its receptor. Further studies show that VEGF hasneuroprotective and neurotrophic role in the stress state of the nerve cells (such as hypoxia, glucose deprivation). This role may be realized through the inhibition of neuronalapoptosis,promotion of neurogenesis and angiogenesis. The existing experiments haveconfirmed that EPCs can secrete VEGF in vitro. However, the problem is whether the EPCstransplantation can play the neuroprotective effect in the cerebral ischemia and what themechanism is. Current research on these issues cannot be reached consensus.Our experiment is based on the fact that EPCs can secrete VEGF in vitro,and observeswhether EPCs can secrete VEGF in the ischemic local brain tissue after building rats tMCAOmodel and transplanting EPCs through the internal carotid artery, then observes whether theEPCs can locate in the cerebral ischemia tissue and secrete VEGF throughimmunohistochemistry, and detects the changes among cortical neuronal apoptosis, VEGFand Caspase-3expression through molecular biology technique in different phases to exploretheir relationship,which is to preliminary clarify the therapeutic effect and mechanism ofEPCs transplanted in tMCAO rats, and provides a theoretical basis for the clinical treatmentof ischemic cerebrovascular diseases.ObjectiveTo observe the protection of EPCs transplantation on tMCAO rats,and to preliminaryexplore the mechanism.Method1Isolating, culturing and identifying EPCs derived from rats bone marrow.2Building rats tMCAO models.3Transplanting EPCs into rats tMCAO models through the internal carotid artery andestablish PBS injection as a control.4Observing the following indicators:①Using stem cells tracer technique to observewhether the EPCs can locate in local brain ischemia tissue;②Using the ordinary HE stainingobserve the pathological changes of the ischemic site;③using the TUNEL method to observethe changes in cortical neuronal apoptosis, and immunohistochemical assay to test thechanges of VEGF and Caspase-3expression,and Western Blot to detect the changes ofVEGF expression in ischemic regional cerebral in respectively1,3,5,7days after transplantation.Results1After induced by VEGF, bFGF,the cells derived from rats bone marrow differentiateand show the following feature:the typical “blood island”-like colony form, the “cobblestone”-like cells, phagocytosing DiI-ac-LDL, being positive in the immunofluorescencestaining for CD34and vWF. It indicates that isolated and cultured cells are EPCs.2EPCs appear red fluorescence in the cytoplasm after phagocytosing DiI-ac-LDL. Theischemia local brain tissues are observed after frozen section for3days when EPCs weretransplanted into rats tMCAO model through internal carotid artery, and the red fluorescentcells can be seen in the vascular wall; no red fluorescent cells can be seen in thecorresponding site in the PBS control group. It indicates that EPCs can locate in theischemia local brain tissues after injected though internal carotid artery.3The influence that EPCs transplanted through internal carotid artery acts on corticalneurons apoptosis of tMCAO rats: Detected by TUNEL assay, the positive cells in theischemic parts in PBS control group gradually increase(P＜0.05), as1d(105.72±2.69),3d(132.96±2.88),5d(145.12±3.22); and they gradually decrease in thetransplantation group(P＜0.05),as1d(91.84±3.21),3d(82.08±2.77),5d(54.68±3.12).Thedifferences in corresponding phase are significant between the two groups(P＜0.05). Itindicates that EPCs can reduce neuron apoptosis of tMCAO rats after injected though internalcarotid artery.4The influence that EPCs transplanted through internal carotid artery acts on the neuronCaspase-3protein expression level of tMCAO rats: Detected by immunohistochemical assay,the positive cells in the ischemic parts in PBS control group gradually increase(P＜0.05), as1d(81.48±3.80),3d(103.00±2.73),5d(122.92±2.65); and they gradually decrease in thetransplantation group(P＜0.05),as1d(71.88±2.55),3d(60.84±3.06),5d(31.24±2.73).Thedifferences in corresponding phase are significant between the two groups(P＜0.05).Itindicates that EPCs can restrain Caspase-3expression of tMCAO rats after injected thoughinternal carotid artery.5The influence that EPCs transplanted through internal carotid artery acts on the VEGFprotein expression level in the ischemic parts of tMCAO rats: Observing whether there is apositive expression of VEGF in the local ischemic brain tissue from morphology byimmunohistochemical assay,VEGF-positive cells are easily detected in the transplantationgroup, but rarely detected in the PBS control group. Further application of the Western Blotassay to observe the change of VEGF protein expression in ischemic regional brain tissue,theamout of VEGF protein expression in PBS control is in low level, as 1d(0.21±0.02),3d(0.34±0.03),5d(0.29±0.05); and it gradually increases in the transplantationgroup(P＜0.05),as1d(0.47±0.02),3d(0.56±0.01),5d(0.99±0.03). There are significantdifferences in corresponding phase between the two groups(P＜0.05). It indicates that EPCscan up-regulate VEGF expression of tMCAO rats in the ischemia local brain tissues afterinjected though internal carotid artery.Conclusions1After induced by VEGF, bFGF,the cells derived from rats bone marrow differentiateand accord with the characteristics of EPCs.2EPCs can locate in the local ischemic brain capillary wall after transplantating into ratstMCAO model through internal carotid artery.3In the EPCs transplantation group, compared with the PBS group, the cortical neuronalapoptosis was decreased gradually, while ischemic local brain tissue VEGF protein expressionwas increased steadily, and neurons Caspase-3protein expression was reduced gradually astime went on after the transplantation. The results suggest that the neuroprotective effectwhich EPCs transplanted through internal carotid artery acting on tMCAO rat brain tissuemay be related to the fact that the paracrine of VEGF inhibits the Caspase-3expression,thereby reducing cortical neurons apoptosis. |
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