骨髓间充质干细胞移植对急性脊髓损伤脱髓鞘病变的保护作用

Protective effect of transplantation of bone mesenchymal stem cells on demyelination in spinal cord injury

骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)已被广泛应用于治疗脊髓损伤,但目前对其治疗机制了解甚少。BMSCs被移植至脊髓钳夹损伤模型大鼠,以研究其保护作用。通过LFB(Luxol fast blue)染色、锇酸染色、TUNEL(Td T-mediated d UTP nick-end labeling)染色和透射电镜对白质有髓神经纤维进行观察。免疫印迹检测BMSCs移植对脑源性神经营养因子(Brain derived neurotrophic factor,BDNF)和caspase 3蛋白表达的影响。通过脊髓损伤后1、7、14 d三个时间点移植BMSCs并进行后肢运动评分(Basso,beattie and bresnahan;BBB评分)和CNPase(2′,3′-cyclic-nucleotide 3′-phosphodiesterase)、髓鞘碱性蛋白(Myelin basic protein,MBP)、caspase 3蛋白水平的检测。免疫荧光观察BMSCs移植到受损脊髓后分化情况及CNPase-caspase 3^+共表达情况。骨髓间充质干细胞移植7 d后,部分移植的BMSCs可表达神经元和少突胶质细胞标记物,大鼠后肢运动能力和髓鞘超微结构特征均明显改善。骨髓间充质干细胞移植后BDNF蛋白表达水平增加,caspase 3蛋白表达水平则降低。相对于脊髓损伤后1 d和14 d,7 d移植BMSCs后MBP和CNPase蛋白表达水平最高;caspase 3蛋白表达水平则最低。骨髓间充质干细胞移植后CNPase-caspase 3^+细胞散在分布于脊髓白质。结果表明,急性脊髓损伤后,BMSCs移植到受损脊髓有分化为神经元和少突胶质细胞的倾向,并促进BDNF的分泌介导抗少突胶质细胞凋亡而对神经脱髓鞘病变有保护作用,且最佳移植时间为脊髓损伤后7 d。

Bone mesenchymal stem cells（BMSCs） have been used worldwide to treat spinal cord injury, but their therapeutic mechanism is poorly understood. In this study, BMSCs were transplanted to aneurysm clip-injured rats to demonstrate their protective effect. We observed myelin sheaths through Luxol fast blue（LFB） staining, osmic acid staining, TUNEL and transmission electron microscopy（TEM）. We performed Western blotting to analyze the expressions of brain-derived neurotrophic factor（BDNF） and caspase 3. BMSCs were transplanted at 1, 7 and 14 days after spinal cord injury. Hindlimb movement（Basso, Beattie and Bresnahan; BBB） score, CNPase（2＇, 3＇-cyclic-nucleotide 3＇-phosphodiesterase）, myelin basic protein（MBP） and caspase 3 protein levels were detected. Immunofluorescence was used to test the differentiation of BMSCs after implanted into damaged spinal cord and co-expression of CNPase-caspase 3＋. At 7 days after BMSCs transplantation, some injected BMSCs expressed neuronal and oligodendrocyte markers. And both locomotor skills and ultra-structural features of myelin sheaths were significantly improved. The expressions of BDNF were clearly increased by BMSCs transplantation, the expression of caspase 3 was the opposite. Compared with the 1 and 14 days transplantation after spinal cord injury, MBP and CNPase expressions were highest, caspase 3 expression was lowest in 7 days BMSCs transplantation. After BMSCs transplantation, CNPase-caspase 3~＋ cells scattered in the white matter of the spinal cord. Therefore, BMSCs had a tendency to differentiate into neurons and oligodendrocytes after transplantation, which could promote the secretion of BDNF. BMSCs protected neural myelin sheaths by inhibiting oligodendrocyte apoptosis via increased secretion of BDNF after SCI. The best therapeutic time was 7 days after spinal cord injury.