骨髓来源神经干细胞对脊髓损伤大鼠运动功能恢复的影响

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目的:探讨骨髓来源神经干细胞对脊髓损伤大鼠运动功能的恢复的影响。方法:分离并体外纯化骨髓间充质干细胞,诱导分化为神经干细胞。60只大鼠按照随机数字表法分为对照组、模型组和治疗组。采用打击器制备脊髓损伤模型,假手术组大鼠仅行椎板切除术暴露硬脊膜,不予打击,直接缝合,给予磷酸盐缓冲液(PBS);模型组大鼠造模后,给予PBS;治疗组大鼠建模后移植骨髓诱导分化的神经干细胞。3组大鼠治疗10 d后,采用Basso、Beattie和Bresnahan(BBB)评分评定3组大鼠后肢功能恢复情况;免疫蛋白印迹法(Western blot)检测各组大鼠脊髓组织中胶质细胞源性神经营养因子(GDNF)、神经生长因子(NGF)、睫状神经营养因子(CNTF)和源性神经营养因子(BDNF)相对表达水平,尼氏染色法检测各组大鼠脊髓尼氏小体的数量,免疫组织化学分析神经丝蛋白200(NF200)阳性的生神经元数量。组间比较采用方差分析,进一步两两比较采用LSD检验。结果:脑脊液诱导分化7 d后的骨髓来源单核细胞Tubulin Ⅲ和胶质纤维酸性蛋白(GFAP)蛋白阳性率[(96.10±12.59)%、(92.16±10.18)%]明显高于脑脊液诱导前骨髓来源单核细胞Tubulin Ⅲ和GFAP蛋白阳性率(0,0),差异有统计学意义(n t=12.028、11.237,n P<0.05)。治疗组大鼠BBB评分[(9.00±2.98)分]明显高于模型组[(3.75±1.09)分],差异有统计学意义(n t=8.195,n P<0.05)。治疗组大鼠脊髓组织中GDNF、NGF、CNTF和BDNF表达水平(1.54±0.21、1.47±0.18、2.89±0.32、1.28±0.17)高于对照组大鼠脊髓组织(0.69±0.11、0.95±0.14、1.32±0.18、0.84±0.13),差异有统计学意义(n t=2.891、2.544、2.758、1.977,n P<0.05)。治疗组大鼠脊髓组织中脊髓组织中尼氏小体和神经元数量[(15.36±4.19)个和(10.24±4.01)个]高于对照组大鼠脊髓组织[(8.10±2.95)个和(5.10±1.16)个],差异有统计学意义(n t=4.823、3.809,n P<0.05)。n 结论:骨髓来源神经干细胞可促进脊髓组织分泌神经营养因子,并修复脊髓神经组织,恢复大鼠运动功能。“,”Objective:To investigate the effect of bone marrow-derived neural stem cells on the recovery of spinal cord injury in rats.Methods:Bone marrow-derived stem cells were isolated and induced to differentiate into neural stem cells n in vitro. The spinal cord injury model was made by percussion apparatus. In the sham operation group, only laminectomy was performed to expose the dura mater without percussion, the incision was directly sutured and phosphate buffer (PBS) was given. Rats in the model group were treated with PBS after modeling. Rats in the treatment group were transplanted with neural stem cells from bone marrow. After 10 d of treatment, Basso, Beattie and Bresnahan (BBB) scores were used to evaluate the functional recovery of hind limbs in the three groups. The relative expression levels of glial cell line derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the spinal cord of rats in three groups were detected by Western blotting. The number of Nissl bodies in the spinal cord of rats in each group was detected by Nissl staining. The number of neurofilament 200 (NF200) positive neurons was analyzed by immunohistochemistry.n Results:The positive rates of tubulin Ⅲ and glial fibrillary acidic protein (GFAP) protein in bone marrow-derived monocytes after 7 d of CSF induction [(96.10±12.59)%, (92.16±10.18)%] were significantly higher than those before CSF induction (0, 0, n t=12.028, 11.237, n P<0.05]. The BBB score in the treatment group (9.00±2.98) was significantly higher than that in the model group (3.75±1.09,n t=8.195, n P<0.05). The expression levels of GDNF, NGF, CNTF and BDNF in spinal cord tissue of treatment group (1.54±0.21, 1.47±0.18, 2.89±0.32, 1.28±0.17) were significantly higher than those of control group (0.69±0.11, 0.95±0.14, 1.32±0.18, 0.84±0.13,n t=2.891, 2.544, 2.758, 1.977, n P<0.05). The number of Nissl bodies and neurons in the spinal cord tissue of the treatment group (15.36±4.19, 10.24±4.01) was significantly greater than that of the control group (8.10±2.95, 5.10±1.16,n t=4.823, 3.809, n P<0.05).n Conclusion:Bone marrow-derived neural stem cells can promote the secretion of neurotrophic factor in spinal cord tissue, repair spinal cord nerve tissue and restore motor function of rats.
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