Himes B T, Liu Y, Solowska J M, Snyder E Y, Fischer I, Tessler A
Department of Neurobiology and Anatomy, MCP Hahnemann University, Philadelphia, Pennsylvania 19129, USA.
J Neurosci Res. 2001 Sep 15;65(6):549-64. doi: 10.1002/jnr.1185.
To test the idea that genetically engineered cells can rescue axotomized neurons, we transplanted fibroblasts and immortalized neural stem cells (NSCs) modified to express neurotrophic factors into the injured spinal cord. The neurotrophin-3 (NT-3) or nerve growth factor (NGF) transgene was introduced into these cells using recombinant retroviral vectors containing an internal ribosome entry site (IRES) sequence and the beta-galactosidase or alkaline phosphatase reporter gene. Bioassay confirmed biological activity of the secreted neurotrophic factors. Clarke's nucleus (CN) axons, which project to the rostral spinal cord and cerebellum, were cut unilaterally in adult rats by T8 hemisection. Rats received transplants of fibroblasts or NSCs genetically modified to express NT-3 or NGF and a reporter gene, only a reporter gene, or no transplant. Two months postoperatively, grafted cells survived at the hemisection site. Grafted fibroblasts and NSCs expressed a reporter gene and immunoreactivity for the NGF or NT-3 transgene. Rats receiving no transplant or a transplant expressing only a reporter gene showed a 30% loss of CN neurons in the L1 segment on the lesioned side. NGF-expressing transplants produced partial rescue compared with hemisection alone. There was no significant neuron loss in rats receiving grafts of either fibroblasts or NSCs engineered to express NT-3. We postulate that NT-3 mediates survival of CN neurons through interaction with trkC receptors, which are expressed on CN neurons. These results support the idea that NT-3 contributes to long-term survival of axotomized CN neurons and show that genetically modified cells rescue axotomized neurons as efficiently as fetal CNS transplants.
为了验证基因工程细胞能否拯救轴突切断的神经元这一想法，我们将经过改造以表达神经营养因子的成纤维细胞和永生化神经干细胞（NSCs）移植到受损的脊髓中。使用含有内部核糖体进入位点（IRES）序列以及β-半乳糖苷酶或碱性磷酸酶报告基因的重组逆转录病毒载体，将神经营养因子-3（NT-3）或神经生长因子（NGF）转基因导入这些细胞。生物测定证实了分泌的神经营养因子的生物活性。在成年大鼠中，通过T8半横切单侧切断投射到脊髓头端和小脑的克拉克核（CN）轴突。大鼠接受了经基因改造以表达NT-3或NGF以及一个报告基因的成纤维细胞或NSCs移植、仅移植一个报告基因或不进行移植。术后两个月，移植的细胞在半横切部位存活。移植的成纤维细胞和NSCs表达报告基因以及对NGF或NT-3转基因的免疫反应性。未接受移植或接受仅表达报告基因的移植的大鼠，在损伤侧L1节段的CN神经元损失了30%。与单纯半横切相比，表达NGF的移植产生了部分拯救效果。接受经基因改造以表达NT-3的成纤维细胞或NSCs移植的大鼠，没有明显的神经元损失。我们推测NT-3通过与CN神经元上表达的trkC受体相互作用来介导CN神经元的存活。这些结果支持了NT-3有助于轴突切断的CN神经元长期存活的观点，并表明基因改造细胞拯救轴突切断的神经元的效率与胎儿中枢神经系统移植相同。
