骨髓间充质干细胞移植对大鼠脊髓损伤后再修复作用的研究

Effects of mesenchymal stem cells transplantation on the repairing of spinal cord injury

目的 探讨间充质干细胞（MSCS）移植对大鼠脊髓损伤后脑源性神经营养因子（BDNF）表达的影响.方法 选取SD大鼠4只作骨髓间充质细胞的分离与培养,提取MSCS;60只大鼠制做脊髓横断损伤模型,细胞移植组24只,PBS（磷酸盐缓冲液）液组24只,空白对照组12只.于脊髓损伤后第7天,无菌条件下,细胞移植组以微量注射器缓慢注入含MSCS的培养液5μl,PBS组注入缓冲液体5 μl,对照组未加任何干预因素.分别于术后7、14、28 d麻醉下行心脏灌流固定取T10节段脊髓,细胞移植组与磷酸盐缓冲组取出损伤节段的脊髓（8只/时点）,空白对照组于同一节段取出相应脊髓（4只/时点）.应用免疫组化法观察MSCS移植后大鼠脊髓损伤区BDNF的表达变化.结果 细胞接种24 h后贴壁生长,72 h细胞增殖,有细胞团形成,在传代过程中,4代以前的细胞倍增时间约为4～6 d,至10代以后细胞增殖能力有所减弱,胞体变得扁平,若加入碱性成纤维细胞生长因子（bFGF）,则可维持基增殖能力和形态.脑源性神经营养因子在正常大鼠脊髓组织中有一定表达,间充质干细胞移植术后第7、14及28天,细胞移植组脑源性神经营养因子均高水平表达,与缓冲液组相比较差异有统计学意义（14 d：0.31±0.03 vs 0.25±0.04,P＜0.01）.结论 间充质干细胞在移植后通过上调脑源性神经营养因子的表达从而促进轴突的再生可能是治疗脊髓损伤的重要机制.

Objective To observe the effects of mesenchymal stem cells （MSCS） transplantation on the brain-derived neurotrophic factors （BDNF） after the spinal cord injury （SCI） of rats, and investigate the mechanism of repairing the SCI by MSCS transplantation.Methods Mesenchymal stem cells were cultured from the thighbone of adult SD rats and identified by immunoctochemistry.Seven days after the operation of spinal cord injury, the mesenchymal stem cells were transplanted into the injured spinal cord site.Sixty adult SD rats were random divided into three groups： Spinal cord injury cured with transplants of mesenchymal stem cells to the injured spinal cord site （ group A）, spinal cord injury received PBS solution（ group B） and control group （ group C）.The expression of brain - derived neurotrophic factors of the lesion and neighbor areas were examined by immunohistochemistry, The mechanism of repairing the lesion and neighbor areas were examined by immunohistochemistry, and the mechanisms of repairing the spinal cord injury after mesenchymal stem cells transplantation were investigated.Results Mesenchymal stem cells began to grow after 24 hours of inoculation and proliferate 72 hours later.The proliferate the proliferous cyclewas 4 ～6 days, it declined on the 10th era.Mesenchymal stem cells turned to flat, and the mesenchymal stem cells could maintain the ability of proliferation if bFGF was added.Compared with group B, transplantation of mesenchymal stem cells enhanced more expression of brain-derived neurotrophic factors in group A （ 14 d ：0.31 ± 0.03 vs 0.25 ± 0.04, P ＜ 0.01 ）.Conclusion Mesenchymal stem cells in transplantation group showed a continued high level of brain-derived neurotrophic factors, which may be one of the mechanisms of repairing the spinal cord injury by mesenchymal stem cells transplantation.