Sayers S T, Khan N, Ahmed Y, Shahid R, Khan T
Department of Veteran Affairs, Edward Hines, Jr. Hospital, IL 60141, USA.
J Mol Neurosci. 1998 Apr;10(2):143-60. doi: 10.1007/BF02737125.
One reason that the central nervous system of adult mammals does not regenerate after injury is that neurotrophic factors are present only in low concentrations in these tissues. Recent studies have shown that the application of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) acts to encourage the regrowth of motor and sensory fibers after spinal cord injury. Other studies have reported that the regrowth of axons after injury was enhanced by the implantation of Schwann cells, which normally secrete BDNF and NT-3. The purpose of the present study was to genetically modify Schwann cells to secrete increased amounts of BDNF or NT-3 by infection with a retroviral vector. Retroviral vectors were constructed by the ligation of BDNF or NT-3 cDNA to the LXSN vector. Viruses were generated from the plasmid forms of the vectors by transient transfection of PA317 amphotrophic retroviral packaging cells. Viruses were harvested and used to infect the human Schwann cell line designated NF-1T. Northern blot analysis of poly (A+) RNA prepared from Schwann cells that were infected with BDNF- or NT-3-containing virus showed the presence of BDNF or NT-3 mRNA. An enzyme-linked immunosorbent assay (ELISA) for BDNF and NT-3 was performed on media the cells were grown in, and on cellular extracts prepared from the BDNF- and NT-3-infected Schwann cells. The ELISA results demonstrated that the Schwann cells were secreting increased levels of immunologically active BDNF or NT-3. Immunocytochemical staining of these cells revealed the presence of these two neurotrophic factors located in perinuclear granules. These neurotrophic factor-secreting Schwann cells are currently being evaluated for their efficacy in the treatment of spinal cord injury.
成年哺乳动物中枢神经系统损伤后不能再生的一个原因是这些组织中神经营养因子的浓度很低。最近的研究表明，应用脑源性神经营养因子（BDNF）和神经营养素-3（NT-3）可促进脊髓损伤后运动和感觉纤维的再生。其他研究报告称，植入通常分泌BDNF和NT-3的雪旺氏细胞可增强损伤后轴突的再生。本研究的目的是通过用逆转录病毒载体感染对雪旺氏细胞进行基因改造，使其分泌更多的BDNF或NT-3。通过将BDNF或NT-3 cDNA连接到LXSN载体上构建逆转录病毒载体。通过瞬时转染PA317嗜性逆转录病毒包装细胞从载体的质粒形式产生病毒。收获病毒并用于感染指定为NF-1T的人雪旺氏细胞系。对用含BDNF或NT-3的病毒感染的雪旺氏细胞制备的聚腺苷酸（A+）RNA进行Northern印迹分析，结果显示存在BDNF或NT-3 mRNA。对细胞生长的培养基以及从感染BDNF和NT-3的雪旺氏细胞制备的细胞提取物进行BDNF和NT-3的酶联免疫吸附测定（ELISA）。ELISA结果表明，雪旺氏细胞分泌的具有免疫活性的BDNF或NT-3水平有所增加。对这些细胞进行免疫细胞化学染色，结果显示这两种神经营养因子存在于核周颗粒中。目前正在评估这些分泌神经营养因子的雪旺氏细胞在治疗脊髓损伤方面的疗效。
