低氧预处理脐带间充质干细胞移植促进大鼠脊髓损伤修复

Transplantation of hypoxia-preconditioned umbilical cord mesenchymal stem cells promotes recovery after spinal cord injury in rat

目的:评估低氧预处理脐带间充质干细胞(human umbilical cord mesenchymal stem cells,UCMSCs)移植修复大鼠急性脊髓损伤的作用并初步探讨其机制。方法:分别于常氧和低氧条件下培养UCMSCs,细胞免疫荧光鉴定细胞,ELISA测定细胞脑源性生长因子(BDNF)、血管源性生长因子(VEGF)、睫状生长因子(CNTF)和肝细胞生长因子(HGF)的浓度。采用改良的Al1en’s法(2.5 g×10 cm)建立60只大鼠T10脊髓损伤模型,随机分为假手术组、模型组、低氧细胞移植组及常氧细胞移植组,每组20只,损伤后即刻移植1×10~6个细胞至低氧细胞移植组及常氧细胞移植组动物脊髓内。术前及术后每周进行运动功能BBB评分;采用免疫荧光法检测移植细胞的存活、神经细胞凋亡;采用ELISA法检测细胞移植第2、14、28 d脊髓组织肝细胞生长因子的浓度。结果:在低氧组,体外由UCMSCs分泌的BDNF、VEGF、CNTF和HGF浓度显著高于常氧组细胞。与模型组比较,细胞移植组运动功能评分和HGF含量增加,而神经细胞的凋亡率降低,但以低氧细胞移植组改变最明显。结论:低氧预处理的UMCSCs具有提高神经营养因子分泌作用,其机制可能与下调神经细胞凋亡,增加受损脊髓组织HGF有关。

Objective： To investigate the effects of transplantation of hypoxia preconditioned umbilical cord mesenchyreal stem cells on the recovery of acute spinal cord injury in rats and explore the mechanisms underlying this process. Methods： Primary cultures growing in normoxic conditions （21% oxygen, N-UCMSC） were either transferred into hy- poxie conditions （5 % 02, H-UCMSC） or continued to grow under standard 21% oxygen conditions for varying time periods. The cells were characterized using immunocytochemistry, the concentration of brain derived neurotrophic factor （ BD- NF）, vascular endothelial growth factor （VEGF） , ciliary neurotrophic factor （CNTF） and hepatoeyte growth factor （HGF） in the conditioned medium were detected by Enzyme-linked immunosorbent assay （ELISA）. Sixty adult rats were subjected to spinal cord injury （SCI） at T10 level using modified Allen＇s method （2.5 g × 10 cm） , SCI rats were ran- domly divided into 3 experimental groups （ twenty rats each group） ： a control group receiving PBS （ control group ） ; a group receiving hypoxia-preconditioned UCMSCs （H-UCMSC group）, and a group receiving normoxic oxygen UCMSCs （ N-UCMSC group） ; a sham group was also included （sham group, n = 20）. 1 × 10^6 N-UCMSCs or H-UCMSCs were injected into the injured spinal cord immediately after SCI. Locomotor recovery was assessed with BBB scoring pre-operation and post-operation weekly; the survival of grafted cells and the apoptosis of neural cells were detected by immunofluorescenee. The expression of HGF on the extracts from the injured spinal cord were examined by ELISA at 2, 14 and 28 days post-injury. Results： In hypoxia group, the concentrations of BDNF, VEGF, CNTF and HGF secreted by UCMSCs in vitro were significantly higher than those in normoxia group. Compared with the model group, the motor function score and HGF content increased in the cell transplantation group, while the apoptosis rate of nerve cells decreased, and the most obvious change was in hypoxia group. Conclusion ： Hypoxie preconditioning with UMCSCs can improve the secretion of neurotrophic factors, and its mechanism may be related to down-regulation of neuronal apoptosis and increase of HGF in injured spinal cord tissue.