内皮祖细胞脊髓内移植对脊髓损伤后神经保护的实验研究

发布时间：2018-05-25 10:38

本文选题：脊髓损伤 + VEGF　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的探索脊髓损伤后内皮祖细胞的脊髓内移植是否为脊髓的恢复提供神经保护作用,为内皮祖细胞移植治疗脊髓损伤的研究提供理论基础。方法从80-100 g SD大鼠双后肢提取骨髓经梯度密度离心后,经诱导培养基培养两周后用细胞形态学和细胞功能学鉴定为内皮祖细胞(Endothelial progenitor cells,EPCs),用CM-Dil染料标记后制成浓度为1×105个/ml细胞悬液备用。用改良Allen法在成年SD大鼠上制造脊髓损伤模型,并立即在损伤部位硬膜下注射EPCs悬液,同时注射EPCs培养基作为对照。对所有大鼠脊髓损伤模型分别在损伤后1、2、3、4、5、6w评估大鼠后肢功能运动评分(BBB score)。分别在1w和2w末取出脊髓,直接在荧光显微镜下观察CM-Dil标记细胞位置。用WB测量脊髓损伤部位VEGF蛋白表达量,免疫组织化学方法检测局部血管数量及血管新生程度、凋亡细胞比例。脊髓损伤6w末用免疫组化方法检测局部神经元和星形胶质细胞数量、HE染色横断面观察脊髓损伤情况。结果(1)脊髓损伤后硬膜下移植EPCs后,EPCs在局部损伤部位可以存活,并且可以迁移到脊髓实质。(2)脊髓损伤1w、2w后EPCs移植组VEGF蛋白表达量明显高于对照组(P0.05)。在2w末EPCs移植后脊髓局部内皮细胞数目比例(38.32±1.49%vs19.02±0.47%,P0.05)和新生血管(16.12±0.79%versus7.61±0.63%,P0.05)较对照组较多,差异有统计学意义(P0.05)。(3)手术2w后实验组大鼠局部脊髓凋亡细胞数量比例明显少于对照组(38.50±5.01%vs 18.67±1.86%,P0.05)。(4)脊髓损伤6w后EPCs移植大鼠损伤部位神经元数量比例(32.04±3.33%vs 19.34±2.70%,P0.05)明显多于对照组大鼠,HE染色观察实验组组织空洞面积小于对照组(27±4.59%vs59±3.27%,P0.05),差异有统计学意义。(5)。随着手术后时间的延长,实验组和对照组大鼠的运动功能均有改善,BBB评分逐渐升高,且在每个时间点实验组大鼠BBB评分稍高于对照组大鼠,在术后第6w EPCs移植大鼠评分达到7.7±1.34,EPCs培养基移植大鼠评分为6.1±1.10,差异有统计学意义(P0.05)。结论脊髓损伤后移植内皮祖细胞可以为损伤局部细胞提供神经保护作用,从而促进运动功能恢复。
[Abstract]:Objective to investigate whether intraspinal transplantation of endothelial progenitor cells after spinal cord injury can provide neuroprotective effect for spinal cord recovery and provide theoretical basis for the treatment of spinal cord injury by transplantation of endothelial progenitor cells. Methods Bone marrow was extracted from 80-100 g SD rats by gradient density centrifugation. Endothelial progenitor cells were identified as Endothelial progenitor cells (EPCs) by cell morphology and cell function after two weeks of culture on the induction medium, and 1 脳 105 / ml cell suspensions were prepared with CM-Dil dye. The spinal cord injury model was established on adult SD rats by modified Allen method. EPCs suspension was injected into the epidural immediately and EPCs medium was injected as control. All the spinal cord injury models in rats were evaluated for functional motor score of hind limbs and BBB scoreboard at 1: 2, 3, 4, 5 and 5 weeks after injury. The spinal cord was removed at the end of 1 and 2 weeks, and the location of CM-Dil labeled cells was observed directly under fluorescence microscope. The expression of VEGF protein in spinal cord injury was measured by WB. The number of local blood vessels, the degree of angiogenesis and the proportion of apoptotic cells were detected by immunohistochemistry. At the end of 6 weeks after spinal cord injury, the number of neurons and astrocytes were detected by immunohistochemical method. Results 1) EPCs could survive and migrate to spinal parenchyma after subdural transplantation of EPCs after spinal cord injury. The expression of VEGF protein in EPCs transplantation group was significantly higher than that in control group (P 0.05) 2 weeks after spinal cord injury. At the end of 2 weeks, the ratio of the number of spinal cord endothelial cells (38.32 卤1.49%vs19.02 卤0.47) and neovascularization (16.12 卤0.79%versus7.61 卤0.63) was higher than that in the control group. The number of apoptotic cells in local spinal cord in experimental group was significantly lower than that in control group (38.50 卤5.01%vs 18.67 卤1.86P 0.05, P 0.05). After 6 weeks of spinal cord injury, the number of neurons in the injured site of EPCs transplanted rats was 32.04 卤3.33%vs 19.34 卤2.70P0.05) significantly higher than that in control group. The tissue cavity area of the experimental group was smaller than that of the control group (27 卤4.59%vs59 卤3.27) by HE staining. The difference was statistically significant. With the extension of the time after operation, the motor function of the experimental group and the control group improved gradually, and the BBB score of the experimental group was slightly higher than that of the control group at each time point. At the 6th week after operation, the score of EPCs transplantation rats reached 7.7 卤1.34.The score of rats transplanted with EPCs medium was 6.1 卤1.10.The difference was statistically significant (P 0.05). Conclusion Endothelial progenitor cells transplanted after spinal cord injury can provide neuroprotective effect for local injured cells and promote the recovery of motor function.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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