Fadeev Filip Olegovich, Bashirov Farid Vagizovich, Markosyan Vahe Arshaluysovich, Izmailov Andrey Alexandrovich, Povysheva Tatyana Vyacheslavovna, Sokolov Mikhail Evgenyevich, Kuznetsov Maxim Sergeevich, Eremeev Anton Alexandrovich, Salafutdinov Ilnur Ildusovich, Rizvanov Albert Anatolyevich, Lee Hyun Joon, Islamov Rustem Robertovich
Department of Medical Biology and Genetics, Kazan State Medical University, Kazan, Russia.
Institute of Fundamental Medicine and Biology, Kazan [Volga Region] Federal University, Kazan, Russia.
Neural Regen Res. 2021 Mar;16(3):550-560. doi: 10.4103/1673-5374.293150.
Despite emerging contemporary biotechnological methods such as gene- and stem cell-based therapy, there are no clinically established therapeutic strategies for neural regeneration after spinal cord injury. Our previous studies have demonstrated that transplantation of genetically engineered human umbilical cord blood mononuclear cells producing three recombinant therapeutic molecules, including vascular endothelial growth factor (VEGF), glial cell-line derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) can improve morpho-functional recovery of injured spinal cord in rats and mini-pigs. To investigate the efficacy of human umbilical cord blood mononuclear cells-mediated triple-gene therapy combined with epidural electrical stimulation in the treatment of spinal cord injury, in this study, rats with moderate spinal cord contusion injury were intrathecally infused with human umbilical cord blood mononuclear cells expressing recombinant genes VEGF165, GDNF, NCAM1 at 4 hours after spinal cord injury. Three days after injury, epidural stimulations were given simultaneously above the lesion site at C5 (to stimulate the cervical network related to forelimb functions) and below the lesion site at L2 (to activate the central pattern generators) every other day for 4 weeks. Rats subjected to the combined treatment showed a limited functional improvement of the knee joint, high preservation of muscle fiber area in tibialis anterior muscle and increased H/M ratio in gastrocnemius muscle 30 days after spinal cord injury. However, beneficial cellular outcomes such as reduced apoptosis and increased sparing of the gray and white matters, and enhanced expression of heat shock and synaptic proteins were found in rats with spinal cord injury subjected to the combined epidural electrical stimulation with gene therapy. This study presents the first proof of principle study of combination of the multisite epidural electrical stimulation with ex vivo triple gene therapy (VEGF, GDNF and NCAM) for treatment of spinal cord injury in rat models. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 2.20.02.18) on February 20, 2018.
尽管出现了当代生物技术方法，如基于基因和干细胞的疗法，但对于脊髓损伤后的神经再生，尚无临床确立的治疗策略。我们之前的研究表明，移植产生三种重组治疗分子（包括血管内皮生长因子（VEGF）、胶质细胞源性神经营养因子（GDNF）和神经细胞黏附分子（NCAM））的基因工程化人脐带血单个核细胞，可以改善大鼠和小型猪脊髓损伤后的形态功能恢复。为了研究人脐带血单个核细胞介导的三基因疗法联合硬膜外电刺激治疗脊髓损伤的疗效，在本研究中，将表达重组基因VEGF165、GDNF、NCAM1的人脐带血单个核细胞在脊髓损伤后4小时经鞘内注入中度脊髓挫伤损伤的大鼠体内。损伤后三天，每隔一天在损伤部位C5上方（以刺激与前肢功能相关的颈神经网）和L2损伤部位下方（以激活中枢模式发生器）同时给予硬膜外刺激，持续4周。接受联合治疗的大鼠在脊髓损伤后30天膝关节功能改善有限，胫骨前肌肌纤维面积高度保留，腓肠肌H/M比值增加。然而，在接受基因疗法联合硬膜外电刺激的脊髓损伤大鼠中，发现了有益的细胞结果，如细胞凋亡减少、灰质和白质保留增加，以及热休克蛋白和突触蛋白表达增强。本研究首次提出了在大鼠模型中联合多部位硬膜外电刺激与体外三基因疗法（VEGF、GDNF和NCAM）治疗脊髓损伤的原理验证研究。动物实验方案于2018年2月20日获得喀山国立医科大学动物护理和使用委员会批准（批准号2.20.02.18）。
