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存活神经元的轴突在损伤造成的恶劣环境中无法再生。该实验将测试新的细胞和分子修复策略是否会促进成熟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，我们制备了逆转录病毒和重组腺病毒构建的神经营养因子，并表明通过基因修饰表达BDNF的成纤维细胞在椎管内移植可促进红毛脊髓轴突的再生，从而有助于运动恢复。我们还使用质粒构建体和重组腺病毒在椎管内注射，将基因注入脊髓和椎管上神经元，可以提高它们在轴突切开术后的存活和再生。在本实验中，我们将使用从胚胎大鼠脊髓分离的多能神经干细胞作为移植。这些细胞具有自我更新、分化为神经元和神经胶质细胞以及基因修饰的能力，因此前景广阔。我们将对这些细胞进行基因修饰，使其表达神经营养因子BDNF和NT3或粘附蛋白L1，并通过质粒注射或腺病毒传递抗凋亡基因Bcl-2。我们提出，通过定量测试，这种组合治疗将提高神经元存活和轴突再生，促进运动和感觉功能的恢复。在第一个系列的实验中，我们将验证将工程神经干细胞移植到单侧颈椎半切病变的想法，将与受伤的宿主和供应因子结合，以拯救被axo切断的脊髓和棘上神经元，促进其轴突的再生并增强恢复。在第二个系列的实验中，我们将测试这样一种观点，即联合治疗的最佳策略将刺激下行通路的再生和脊髓横断后后肢功能的恢复，这是一种人类完全脊髓损伤的模型，其中解剖和行为研究的结果是明确的。这些实验结果将有助于开发一种有效的策略来促进神经元存活和轴突再生，从而增强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.