| ObjectiveTo observe the dynamic state changes of body growth rate, neurological impairment,cerebral infarct volume, pathological change of cerebral tissue, microvascular structure, survival of cells, expression of vascular endothelial growth factor(VEGF) and vascular endothelial cell marker CD34 at different intervals after cerebral ischemic reperfusion in operated rats. To exam their feature of brain tissue injury angiogenesis after cerebral ischemic-reperfu sion and reveal its changeable regularity.To study the influence of these change by using BMSCs (bone mesenchymal stem cells, BMSCs) transplantation, VEGF gene-modified BMSCs (BMSCs/VEGF) transplantation, Naomaiyihao (NMYH) and their associated transplantation(NMYH+BMSCs, NMYH+BMSCs/VEGF), respectively. Disc-uss the protective effect and promotion of angiogenesis, to reveal the ther-apeutic mechanism of Naomaiyihao combined BMSCs transplantation to rats after ischemi c-reperfusion.MethodsSD(Sprague Dawley, SD) (n=235, male, weight:250-330g)rats were randomly divided into sham-operated group(n=8) and operated group which involved model group(Model group), BMSCs transplantation group(BMSCs group), VEGF gene-modified BMSCs transplantation group (BMSCs/VEGF group), Naomaiyihao group(NMYH group), and Combined treated group(NMYH+BMSCs group and NMYH+BMSCs/VEGF group), then operated groups were randomly divided into 7d,14d and 21d. Each group had 8,8,11 rats, respectively. Bone mesenchymal stem cells were isolated and cultured from bone marrow by the holo-bone marrow adherence, and were identified according to morphology and surface markers analsis. BMSCs were labeled in vitro by CM-Dil.The 24-hour,21-day and 30-day labeled cells were observed under fluorescence inverted microscope. Growth curve were delineated so as to identify whether or not the marker casted an influence on the growth characteristics in vitra. BMSCs were transfected with eukaryotic expression plasmid pEGFP-VEGF165 by positive ionic liposome transfection method. Expression of pEGFP-VEGF165 fusion protein was detected according fluorescent microscope, Flow cytomertry, enzyme linked immunosorbent assay(ELISA)and RT-PCR method, respectively. Effect of gene transfection on cell proliferation was detected by MTT method. Iscliemic-perfusion animal model was duplicated in accordance with improved Longa's focal middle cerebral artery occlusion(MCAO) model in rats, After 2 hours of ischemia, the fishing line was picked out 3-4mm to make the brain reperfused, and 10μl BMSCs suspension(5×107/ml) were injected respectively by stereotaxic apparatus into striatum in rats of BMSCs group and NMYH+BMSCs group 24 hours after reperfusion. BMSCs/VEGF group and NMYH+BMSCs/VEGF group received equivalent VEGF gene-modified BMSCs in the same way. Model group and NMYH group with serum-free L-DMEM of equal volume.NMYH group, NMYH+BMSCs group and NMYH+BMSCs/VEGF group were fed with Naomaiyihao(1.5g/kg/d) by intragastric administration after transplantation, BMSCs group and BMSCs/VEGF group were fed with equivalent saline by intragastric administration. The body growth rate and neuroethology function of rats were tested on 7d,14d and 21d. Cerebral infarct volume was determined using TTC coloration. Pathological changes in ischemic area was observed from light microscope. Laser scanning confocal microscopy(LSCM) was used to measure angiogenesis. The transplanted CM-Dil labeled BMSCs were detected in brain tissue cryosections by fluorescence microscope. Immunohist-ochemical method was used to detected the expression of VEGF and CD34 in the ischemic boundary zone (IBZ).Result1. BMSCs were adherent cultured and gained uniform shape and similar growth pattern. The expression of CD34, CD44 and CD29 were 1.71%,92.10% and 95.32% respectively on BMSCs. In vitro, the CM-Dil labeled BMSCs displayed red fluorescence,cell labeling rate was 100% at 24 hours. Fluorescence intensity at 21 days was close to 24 hours, but it had became attenuated 30 days after labeling. The growth, proliferation and morphology of BMSCs did not change after CM-Dil labeling. Compared with the model group, numbers of CD34 positive cell significantly increased in BMSCs group, BMSCs/VEGF group and NMYH group. Treatment with NMYH+BMSCs or NMYH+BMSCs/VEGF significantly increased numbers of CD34 positive cell compared with numbers that treated with singleness (P<0.05).Conclusion1.High pured BMSCs can be obtained by the holo-bone marrow adherence. The procedure of CM-Dil labeling is simple, efficiency and noncytotoxic. The minimum time of labeling is 21 days. CM-Dil is available for targeted in brain tracing of rat bone marrow mesenchymal stem cell directional transplantation.2. It is successful to transfected BMSCs with eukaryotic expression plasmid pEGFP-VEGF165.3.Cerebral ischemia reperfusion could result in serious pathological injury. Naomaiyihao, BMSCs transplantation, gene-modified BMSCs transplantat-ion or their combination can resist cerebral ischemic reperfusion injury and improve the neurological function of cerebral ischemic rats, The combinaton NMYH+BMSCs/VEGF has an optimal effect. Enhancement of angiogenesis may be one of the mechanisms of brain protection; Survival, distribution and migration trending to boundary regions of ischemia were observed under the fluorescence microscope after transplantation. Survival of gene-modified BMSCs was the same as BMSCs. Naomaoyihao can increase blood supply around the transplanted cell region, and decrease infarct volume, so it could promote BMSCs survived, differentiated into vascular endothelial cell and the neovascularization in ischemic brain. Mechanism of angiogenesis may be correlated to the up-regulation of expression of VEGF.
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