内皮祖细胞移植对动脉粥样硬化模型大鼠脑缺血再灌注后学习记忆能力与脑顶叶皮质的影响

Effects of transplantation with bone marrow-derived endothelial progenitor cells on learning,memory and neurons in the cortex of the parietal lobe after cerebral ischemia reperfusion injury of atherosclerotic model rats

目的探讨内皮祖细胞(EPCs)移植对动脉粥样硬化(AS)模型大鼠脑缺血再灌注损伤(IRI)后学习记忆能力与脑顶叶皮质结构的影响。方法高脂膳食饲养建立30只动脉粥样硬化大鼠模型,随机分为AS组,IRI组和EPCs移植组。采集骨髓分离EPCs并体外扩增培养,检测其表面标记物的表达;第7天采用线栓法制作局灶性IRI模型,建模成功后1d EPCs移植组经尾静脉移植EPCs,IRI组与AS组给予等量体积的磷酸盐缓冲液。移植后7d检测各组大鼠的行为能力、脑组织血管内皮生长因子(VEGF)含量及其mRNA表达与其结构的病理改变。结果培养24h后见细胞贴壁生长逐渐变为梭形;第3天细胞明显增殖集落形成;第5天细胞集落逐渐增大呈现克隆样生长;第7天细胞汇合达80%;第10～14天细胞基本铺满瓶底呈铺路石样密集排列。荧光显微镜下,DIL-ac-LDL和FITC-UEA-1双荧光染色的细胞数占贴壁细胞数的75%以上。与IRI组相比,EPCs移植后大鼠的学习记忆能力较IRI组明显改善,VEGF含量及其mRNA表达显著下降(P<0.05)。光镜下,EPCs移植组大鼠脑缺血侧顶叶皮质Caspase-3和胶质细胞原纤维酸性蛋白(GFAP)阳性神经元均较IRI组明显下降(P<0.05)。结论 EPCs移植能改善AS模型大鼠脑IRI后的学习记忆能力、减轻脑组织的病理损害,这些变化提示EPCs促进了神经的修复。

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 thromhus. 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 afterseven 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. 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 ceils. 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 （P 〈0.05）, the quantity of Caspase-3 and GFAP immunopositive neurons were obviously decreased in the EPCs transplant group （P 〈 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.