激活态雪旺细胞联合脐血间充质干细胞移植修复脊髓损伤的研究

Activated Schwann cells combined with umbilical cord blood mesenchymal stem cell transplantation for repair of spinal cord injury

目的观察激活态雪旺细胞（ASCs）与人脐血间充质干细胞（HUCBMSCs）局部联合移植后脊髓损伤局部神经标志物的变化，探讨ASCs与HUCBMSCs联合移植在促进大鼠脊髓损伤后轴突再生和功能恢复方面所起的作用。方法采用前期实验方法获得ASCs及HUCBMSCs，并制作成年雌性Wistar大鼠胸10脊髓损伤模型。将80只大鼠随机分成4组：空白对照组[注射杜尔伯科改良伊格尔培养基（DMEM）]；ASCs移植组；HUCBMSCs移植组；ASCs与HUCBMSCs联合移植组。分别将DMEM、ASCs、HUCBMSCs以及ASCs＋HUCBMSCs移植入对应组的脊髓损伤部位。术后采用Basso，Beattie和Bresnahan （BBB）评分对大鼠的后肢功能恢复情况进行评价。于脊髓损伤后1、2、3、4周取脊髓损伤中心组织进行Western blot半定量检测及实时荧光定量聚合酶链反应（FQ-PCR）检测。12周后，从每组随机选取8只大鼠进行10%生物素葡聚糖胺（BDA）顺行示踪标记。标记2周后处死大鼠，将损伤段脊髓取出作5 μm快速冰冻切片后，进行BDA显影、苏木素-伊红（HE）染色，神经丝蛋白-200（NF-200）、胶质纤维酸性蛋白（GFAP）、髓鞘碱性蛋白（MBP）免疫组织化学染色。综合以上数据并进行统计学分析。结果Western blot及FQ-PCR结果显示NF-200及MBP表达量在各组均有升高，但以联合移植组最高（损伤后4周NF-200：4.581±0.588，MBP：3.922±0.259）；损伤后8周，共移植组大鼠的BBB评分[（16.800 0±0.223 0）分]显著高于其余3组（P＝0.000）。BDA顺行示踪标记显示共移植组较ASCs移植组和HUCBMSCs移植组有较多的再生神经纤维通过损伤区，而空白对照组几乎未见再生神经纤维穿越。HE染色显示细胞共移植组损伤空洞明显小于其余3组（P＝0.000）。结论ASCs联合HUCBMSCs移植治疗脊髓损伤可促进更多的HUCBMSCs向神经元方向分化，促进轴突再生，改善脊髓损伤后功能的恢复。

ObjectiveTo observe the variation of neuronal markers after co-transplantation of activated Schwann cells （ASCs） and human umbilical cord blood mesenchymal stem cells （HUCBMSCs）into rat spinal cord injury （SCI）. Also, we discuss whether these two types of cells grafted into the injured site of spinal cord have the ability of promoting axonal regeneration and functional recovery after SCI.MethodsSCI model in Wistar rat （Female, 8 weeks’ old） was created at T10 segment with NYU Impactor-Ⅱmachine of 10 g×50 mm weight drop.80 SCI rats were classified into 4 groups randomly： blank control group, ASCs transplantation group, HUCBMSCs transplantation group and co-transplantation group of ASCs and HUCBMSCs. 1, 2, 3, 4 weeks after operation, 3 animals of each group were sacrificed, and injured spinal cord tissue were used for Western blotting and Real-time polymerase chain reaction analysis [nuclear factor （NF）-200, glial fibrillary acidic protein （GFAP）, myelin basic protein （MBP）]. Basso, Beattie, and Bresnahan （BBB） score was carried out at the 3rd day and at weekly after intervals injury.12 weeks later, 8 rats in each group were selected to carry out 10% BDA anterograde tracing mark.2 weeks later, the animals were sacrificed, the spinal cord tissue with injured lesion was taken out and fast cryostat sections （5 μm） was made followed by BDA developing, hematoxylin and eosin （HE） and immunochemistry （NF-200, MBP, GFAP） staining.ResultsWestern blotting and Real-time PCR analysis revealed that NF-200 and MBP expressed higher with time while the co-graft group is the highest （4 weeks after injury, NF-200： 4.581±0.588, MBP： 3.922±0.259）. From the 4th week after cell-transplantation, BBB score in co-graft group was significantly higher than that in other 3 groups （P=0.000）. The rats of co-graft group recoverd to the largest extent. BDA anterograde injured site in co-graft group than that in other 3 groups, while little in control group （P=0.000）. HE staining showed that the injured cavity was the smallest one in cell co-graft group when compared with other 3 groups.ConclusionThe results indicate that the co-transplantation fully takes advantage of ASCs to promote HUCBMSCs differentiate into neurons. This is more effective than single cell transplantation in promoting axonal regeneration and functional recovery after rat’s spinal cord injury.