CELLULAR AND MOLECULAR STRATEGIES IN SPINAL CORD REPAIR

脊髓损伤的破坏性影响是由于神经元的死亡和幸存神经元的轴突未能通过损伤造成的不适宜居住的环境再生所致。拟议中的实验将测试新的细胞和分子修复策略是否将促进成年大鼠脊髓损伤模型的再生，从而导致运动和感觉恢复。在初步研究中，我们已经制备了神经营养因子的逆转录病毒和重组腺病毒载体，并表明脊髓内移植表达BDNF的成纤维细胞可以促进红核脊髓轴突的再生，这有助于运动恢复。我们还使用了椎管内注射质粒构建体和重组腺病毒来将基因注入脊髓和脊髓上神经元，以增强它们在轴突切断后的存活和再生。在本实验中，我们将使用从胚胎大鼠脊髓分离的多潜能神经干细胞作为移植细胞。这些细胞非常有前途，因为它们具有自我更新、分化为神经元和胶质细胞以及基因修饰的能力。我们将对这些细胞进行基因修饰，以表达神经营养因子BDNF和NT3或黏附蛋白L1，并通过质粒注射或腺病毒携带抗凋亡基因Bcl-2。我们认为，这种联合治疗将增强神经元存活和轴突再生，并促进定量测试所测量的运动和感觉功能的恢复。在第一系列实验中，我们将测试这样一种想法，即将工程化神经干细胞移植到单侧颈椎半横断损伤处，将与受伤的宿主整合，并提供因子，以挽救被切断的脊髓和脊髓上的神经元，促进其轴突的再生和促进恢复。在第二系列实验中，我们将检验综合治疗的最佳策略将刺激脊髓横断后下行通路的再生和后肢功能的恢复，这是一个解剖学和行为学研究结果明确的人类完全性脊髓损伤模型。这些实验结果将有助于开发一种促进神经元存活和轴突再生的有效策略，以促进脊髓损伤后的功能恢复。

The devastating effects of spinal cord injury are due to the death of neurons and to the failure of the axons of surviving neurons to regenerate through the inhospitable environment created by the injury. The proposed experiments will test whether novel cellular and molecular strategies of repair will promote regeneration leading to locomotor and sensory recovery in well-characterized models of spinal cord injury in adult rats. In preliminary studies we have prepared retrovirus and recombinant adenovirus constructs of neurotrophins and shown that intraspinal transplants of fibroblasts genetically modified to express BDNF promote regeneration of rubrospinal axons that contribute to locomotor recovery. We have also used intraspinal injections of plasmid constructs and recombinant adenovirus to administer genes to spinal and supraspinal neurons that can enhance their survival and regeneration after axotomy. In the present experiments we will use as transplants multipotential neural stem cells isolated from embryonic rat spinal cord. These cells are very promising because of their capacities for self- renewal, differentiation into neurons and glia and genetic modification. We will genetically modify these cells to express neurotrophin factors BDNF and NT3 or adhesion protein L1, and in addition deliver the antiapoptotic gene Bcl-2 by plasmid injections or adenovirus. We propose that this combination of treatments will enhance neuron survival and axon regeneration and promote the recovery of locomotor and sensory function as measured by quantitative tests. In the first series of experiments we will test the idea that engineered neural stem cells transplanted into a unilateral cervical hemisection lesion will integrate with the injured host and supply factors that will rescue axotomized spinal and supraspinal neurons, promote regeneration of their axons and enhance recovery. In the second series of experiments we will test the idea that the best strategy of combined treatments will stimulate regeneration of descending pathways and recovery of hindlimb function after spinal cord transection, a model for complete spinal cord injury in humans in which results of anatomical and behavioral studies are unambiguous. The results of these experiments will contribute to developing an effective strategy for promoting neuron survival and axon regeneration that will enhance functional recovery after spinal cord injury.