Cejas P J, Martinez M, Karmally S, McKillop M, McKillop J, Plunkett J A, Oudega M, Eaton M J
The Miami Project to Cure Paralysis, University of Miami School of Medicine, FL 33136, USA.
Pain. 2000 May;86(1-2):195-210. doi: 10.1016/s0304-3959(00)00245-1.
Chronic delivery of anti-nociceptive molecules by means of cell grafts near the pain processing centers of the spinal cord is a newly developing technique for the treatment of neuropathic pain. The rat neuronal cell line, RN33B, derived from E13 rat brainstem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat brain-derived neurotrophic factor cDNA (BDNF), and the BDNF-synthesizing cell line, 33BDNF.4, was isolated. The 33BDNF.4 cells synthesized mature BDNF protein at permissive temperature (33 degrees C), when the cells were proliferating, and during differentiation at non-permissive temperature (39 degrees C) in vitro. The bio-active BDNF protein was also secreted by the cells during both growth conditions, as measured by ELISA analysis of BDNF content and secretion. The bio-activity of the BDNF in 33BDNF.4 cell conditioned media was assessed by neurite outgrowth from E15 dorsal root ganglion (DRG) cultures. A control cell line, 33V1, transfected with the vector alone, did not synthesize or secrete any significant BDNF at either growth condition. Both cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hindpaw. When 33BDNF.4 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord and the 33BDNF.4 cells continued to synthesize BDNF in vivo. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2-7 week period after grafts of 33BDNF.4 cells. The maximal effect on chronic pain behaviors with the BDNF grafts occurred 2-3 weeks after transplant and the anti-nociceptive effects of the BDNF cell grafts was permanent. Transplants of the control 33V1 cells had no effect on the allodynia and hyperalgesia induced by CCI and these cells did not synthesize BDNF in vivo. These data suggest that a chronically applied, low local dose of BDNF supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver anti-nociceptive molecules, such as BDNF, in a model of chronic pain offers a novel approach to pain management and such 'biologic minipumps' can be developed for safe use in humans.
通过在脊髓疼痛处理中心附近进行细胞移植来长期递送抗伤害性分子，是一种新兴的治疗神经性疼痛的技术。大鼠神经元细胞系RN33B源自E13大鼠脑干中缝核，并用大T抗原的SV40温度敏感等位基因（tsTag）永生化，将其用大鼠脑源性神经营养因子cDNA（BDNF）转染，分离出合成BDNF的细胞系33BDNF.4。33BDNF.4细胞在允许温度（33℃）下，即细胞增殖时，以及在非允许温度（39℃）体外分化过程中合成成熟的BDNF蛋白。通过ELISA分析BDNF含量和分泌情况可知，在两种生长条件下细胞均分泌具有生物活性的BDNF蛋白。通过E15背根神经节（DRG）培养物的神经突生长来评估33BDNF.4细胞条件培养基中BDNF的生物活性。仅用载体转染的对照细胞系33V1在两种生长条件下均不合成或分泌任何显著量的BDNF。两种细胞系都被用作坐骨神经单侧慢性压迫损伤（CCI）诱导的慢性神经性疼痛模型中的移植物。在CCI后，对患侧后爪的疼痛相关行为进行评估，包括冷和触觉异常性疼痛以及热和触觉痛觉过敏。当在CCI后1周将33BDNF.4和33V1细胞移植到脊髓腰段蛛网膜下腔时，它们在脊髓周围的软脑膜上存活超过7周，并且33BDNF.4细胞在体内继续合成BDNF。此外，在移植33BDNF.4细胞后的2 - 7周内，CCI诱导的触觉和冷异常性疼痛以及触觉和热痛觉过敏显著减轻。BDNF移植物对慢性疼痛行为的最大作用在移植后2 - 3周出现，并且BDNF细胞移植物的抗伤害性作用是永久性的。对照33V1细胞的移植对CCI诱导的异常性疼痛和痛觉过敏没有影响，并且这些细胞在体内不合成BDNF。这些数据表明，由脊髓背角附近移植细胞提供长期应用的低局部剂量BDNF能够逆转CCI后慢性神经性疼痛的发展。在慢性疼痛模型中使用能够递送抗伤害性分子（如BDNF）的神经细胞系为疼痛管理提供了一种新方法，并且这种“生物微型泵”可开发用于人类安全使用。
