骨髓间充质干细胞立体定向移植治疗大鼠脊髓损伤*★

摘要 背景：传统观念认为，神经组织损伤后几乎不能再生，以往对SCI的治疗缺乏有效手段，致使本病致残率高，疗效差。干细胞治疗关键在于移植具有再生能力的干细胞，通过多种作用机制，可以重建中枢神经系统的结构和功能，近年来引起了广泛的关注。 目的：探讨立体定向移植骨髓间充质干细胞（MSCs）对大鼠脊髓损伤修复的影响并探讨其机制 设计、时间及地点：随机对照动物实验，于2007-10/2008-6在天津市环湖医院完成。 材料：1月龄SD大鼠20只，用于制备骨髓间充质干细胞；健康成年Wistar大鼠45只，雌性、同系，体质量280±20 g。将动物随机分为对照组、假手术组与移植组，每组各15只。 方法：密度梯度离心法结合贴壁筛选法分离骨髓间充质干细胞，经流式细胞仪鉴定为MSCs。以动脉瘤夹夹闭法制备大鼠脊髓损伤（SCI）模型，在SCI大鼠致伤后第7天，通过立体定向途径移植MSCs到移植组大鼠脊髓损伤中心，移植等量生理盐水至假手术组大鼠脊髓损伤中心，对照组大鼠不做处理。 主要观察指标：SCI大鼠损伤前及损伤后第7天、14天、30天、60天、90天的BBB评分；损伤后第90天处死大鼠，观察其脊髓组织中有无BrdU阳性细胞、Brdu+NSE、Brdu+GFAP、Brdu+bFGF、Brdu+BDNF免疫组化双染阳性细胞并观察NSE、GFAP、bFGF、BDNF单染阳性细胞。 结果： ①BBB评分发现，MSCs移植组大鼠BBB后肢功能评分恢复优于对照组（p<0.05）；假手术组BBB评分在损伤后30天内恢复速度慢于对照组（p<0.05），至第90天与对照组比较无显著差异（P>0.05）；②免疫组织化学染色发现，移植组大鼠脊髓内在损伤中心及头、尾端距离脊髓损伤中心1cm处均可见BrdU染色阳性细胞及Brdu+NSE、Brdu+GFAP、Brdu+bFGF、Brdu+BDNF免疫组化双染阳性细胞。移植组NSE、GFAP、bFGF、BDNF单染阳性细胞数明显高于对照组和假手术组（p<0.05）。 结论： MSCs移植可以促进SCI大鼠的神经功能的恢复，其机制可能与移植细胞分化为神经元样和神经胶质细胞样细胞，并分泌或促进宿主分泌神经营养因子有关。 关键词 脊髓损伤 骨髓间充质干细胞 立体定向 细胞移植

Bone mesenchymal stem cell stereotaxis transplantation for the treatment of spinal cord injury in rats

BACKGROUND: The key of stem cells for treating nervous tissue injury is the transplantation of stem cells that have regeneration capacity. The structure and function of central nervous system were re-established by multiple action mechanisms. OBJECTIVE: To explore the effects and mechanisms of bone marrow mesenchymal stem cells (BMSCs) locally transplanted into rats with spinal cord injury on neurological recovery. METHODS: BMSCs were separated with density gradient centrifugation and cell attachment. 10 mg/L BrdU was used for labeling before cell transplantation. Adult female Wistar rats were used to establish spinal cord injury models using an aneurysm clip, and they were then randomly divided into control group, saline group and transplantation group. In the transplantation group, BMSCs were transplanted into the damaged spinal cord by stereotaxis at day 7 following damage. In the saline group, an equal volume of saline was utilized. In the control group, the rats were left intact. Basso, Beattie and Bresnahan (BBB) locomotor rating scale was used before and at 7, 14, 30, 60 and 90 days following damage. Rats were sacrificed at day 90. BrdU-positive cells, Brdu+neuron specific enolase, Brdu+glial fibrillary acidic protein (GFAP), Brdu+basic fibroblast growth factor (bFGF), and Brdu+brain-derived nerve growth factor (BDNF) immunohistochemistry double-staining cells and simple staining positive cells were observed. RESULTS AND CONCLUSION: The recovery of BBB function score was better in the transplantation group than in the control group (P < 0.05). The recovery speed of BBB function score was slower in the saline group than in the control group at 30 days following damage (P < 0.05). No significant difference was determined at day 90 compared with the control group (P > 0.05). BrdU-positive cells and double-staining cells of immunohistochemistry could be found at the center of damage site and 1 cm from caudal end to damaged site in rats of the transplantation group. The number of NSE, GFAP, bFGF and BDNF simple staining cells was significantly greater in the transplantation group than in the control and saline groups (P < 0.05). Results indicated that BMSC transplantation can improve the recovery of nervous function of rats with spinal cord injury. Its mechanism may be correlated with the differentiation of transplanted cells into neuron-like and glial cell-like cells, secretion or promoting secretion of neurotrophic factors in host.