不同时相点移植的皮肤源性前体细胞在损伤脊髓中的存活数量及其机制

Survival rates of skin-derived precursors in injured spinal cord after different time points of transplantation and its mechanisms

目的:观察不同时相点移植的皮肤源性前体细胞(skin-derived precursors,SKPs)在损伤脊髓中的存活数量并探讨其可能的机制。方法:分离雄性大鼠SKPs,Allen法制备雌性大鼠中度脊髓挫伤模型,伤后即刻、1d、3d、7d和14d取损伤脊髓匀浆离心取上清液,酶联免疫吸附法测定上清液中白介素-1(IL-1)和血小板源性生长因子(platelet-derived growth factor,PDGF)的含量,然后观察上清液、IL-1和PDGF对SKPs增殖和凋亡的影响。上述同样的时相点将DAPI标记的SKPs经局部注射到损伤脊髓处,移植后14d取材采用针对Y染色体的实时定量PCR检测脊髓中SKPs含量,BBB法评价大鼠功能恢复情况。结果:IL-1可抑制SKPs的增殖活性,促进其凋亡;而PDGF的作用相反。损伤3d后脊髓的提取液培养中IL-1的浓度最高,7d的PDGF浓度最高,7d的脊髓提取液对SKPs促增殖活性最高。伤后7d移植组损伤脊髓局部存活的SKPs数量最多,功能恢复也最好。结论:中度挫伤脊髓损伤后7d移植SKPs存活率最高,对功能恢复的作用最强,其可能机制与损伤脊髓局部表达的细胞因子有关。

Objective To observe the survival rates of skin-derived precursors （SKPs） in injured spinal cord after different time points of transplantation and to investigate the possible mechanisms. Methods SKPs were isolated from male rats and moderate contusion spinal cord injury （SCI） models were developed by Allen method. Supemant was obtained from injured spinal cord by homogenation and centrifugation immediately, 1 d, 3 d, 7 d, and 14 d after the injury. Then the concentrations of interleukin-1 （IL-1） and platelet-defived growth factor （PDGF） were determined by ELISA. The effects of supemant, IL-1, and PDGF on the proliferation capacity and apoptosis of SKPs were assessed. DAPI labeled SKPs were transplanted into injured spinal cord by local injection at the same time points. Fourteen days after transplantation, real-time PCR was employed to examine the amount of SKPs in injured spinal cord and BBB method was used to assess the functional recovery of rats. Results IL-l inhibited the proliferation capacity and enhanced the apoptosis of SKPs, while PDGF had an opposite effect. The concentration of IL-1 reached the highest in the supernant from the spinal cord 3 days after injury, while the concentration of PDGF reached the highest in supemant from the spinal cord 7 days after injury. The supemant from spinal cord 7 days after injury showed the most potent effect to enhance the proliferation capacity of SKPs. The amount of SKPs was the highest in rats that received transplantation at 7 days after injury, and resulted in best functional recovery. Conclusions In moderate contusion SCI rat models, the survival rate of SKPs was the highest in that received transplantation at 7 days after injury, and resulted in best functional recovery. The possible mechanisms lied in the expressions of cytokines in the sites of SCI.