关键词: 内皮祖细胞, 动脉粥样硬化, 脑缺血, 血管内皮生长因子, 神经元, 免疫组织化学技术, 反转录聚合酶链式反应, 大鼠

Abstract: Objective To study behavior abilities and morphological changes on neurons in the cortex of parietal lobe after cerebral ischemia reperfusion injury (IRI) of atherosclerotic(AS) model rats and observe the effect of transplantation with bone marrow-derived endothelial progenitor cells (EPCs) on the AS model rat. Methods A total of thirty male adult Wister AS model rats were established by fat-rich diet feeding for six consecutive weeks. EPCs were obtained from the bone marrow and the cells cultured in vitro in M199.On the 7th day, middle cerebral artery occlusion (MCAO) rat models were established by the method of thread thrombus. One day after MCAO, the rats were randomly divided into the EPCs transplant group (the EPCs were injected into the caudal vein), the AS group and the IRI group (the same volume of PBS was injected into the caudal vein).The learning and memory abilities were detected by they-maze after seven days. Then, the expression of vascular endothelial growth factor (VEGF) mRNA was detected by reverse transcription polymerase chain reaction and the ELISA method was used to detect the VEGF content. Caspase-3 and GFAP immunopositive cells in the cortex of the parietal lobe were observed under a light microscope, and quantitative analysis was performed by cell morphometric technique. BR>Results EPCs from bone marrow were isolated, induced and cultured successfully in vitro . Following culture for 24 hours, adherent cells presented spindle-shaped appearance. Cell colony-forming units appeared 72 hours after seeding and increased obviously after five days. One week later the cells confluenced to 80%. Attached cells formed a cobblestone-like structure by 10-14 days. Observation using fluorescence microscopy, the double-positive staining with DIL-ac-LDL and FITC-UEA-1 cell population was above 75% among adherent cells. Compared with the IRI group, the learning and memory abilities of the EPCs transplant group were obviously improved, but the content and mRNA of VEGF were significantly decreased (EM>P/EM> <0.05),the quantity of Caspase-3 and GFAP immunopositive neurons were obviously decreased in the EPCs transplant group (EM>P/EM> <0.05). Conclusion EPCs from bone marrow efficiently promote neurological functional recovery and decrease the pathological lesion of the cortex in the parietal lobe after cerebral IRI of AS model rats, which may improve the neovascularization, reduce infarct area and improve neurocrine function. BR>

Key words: Endothelial progenitor cell, Atherosclerosis, Cerebral ischemia, VEGF, Neuron, Immunohistochemical techniques, RT-PCR, Rats