Leaver S G, Cui Q, Plant G W, Arulpragasam A, Hisheh S, Verhaagen J, Harvey A R
School of Anatomy and Human Biology, The University of Western Australia, Western Australia, Australia.
Gene Ther. 2006 Sep;13(18):1328-41. doi: 10.1038/sj.gt.3302791. Epub 2006 May 18.
We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal ganglion cell (RGC) survival and regeneration following (i) optic nerve (ON) crush or (ii) after ON cut and attachment of a peripheral nerve (PN). At 7 weeks after ON crush, quantification of betaIII-tubulin immunostaining revealed that, compared to AAV-GFP controls, RGC survival was not enhanced by AAV-GAP43-GFP but was increased in AAV-CNTF-GFP (mean RGCs/retina: 17 450+/-358 s.e.m.) and AAV-BDNF-GFP injected eyes (10 200+/-4064 RGCs/retina). Consistent with increased RGC viability in AAV-CNTF-GFP and AAV-BDNF-GFP injected eyes, these animals possessed many betaIII-tubulin- and GFP-positive fibres proximal to the ON crush. However, only in the AAV-CNTF-GFP group were regenerating RGC axons seen in distal ON (1135+/-367 axons/nerve, 0.5 mm post-crush), some reaching the optic chiasm. RGCs were immunoreactive for CNTF and quantitative RT-PCR revealed a substantial increase in CNTF mRNA expression in retinas transduced with AAV-CNTF-GFP. The combination of AAV-CNTF-GFP transduction of RGCs with autologous PN-ON transplantation resulted in even greater RGC survival and regeneration. At 7 weeks after PN transplantation there were 27 954 (+/-2833) surviving RGCs/retina, about 25% of the adult RGC population. Of these, 13 352 (+/-1868) RGCs/retina were retrogradely labelled after fluorogold injections into PN grafts. In summary, AAV-mediated expression of CNTF promotes long-term survival and regeneration of injured adult RGCs, effects that are substantially enhanced by combining gene and cell-based therapies/interventions.
我们比较了玻璃体内注射编码增强型绿色荧光蛋白（GFP）以及睫状神经营养因子（CNTF）、脑源性神经营养因子（BDNF）或生长相关蛋白43（GAP43）的双顺反子腺相关病毒（AAV - 2）载体，对成年视网膜神经节细胞（RGC）在以下两种情况下存活和再生的影响：（i）视神经（ON）挤压后；（ii）ON切断并连接外周神经（PN）后。在ON挤压7周后，对βIII - 微管蛋白免疫染色进行定量分析发现，与AAV - GFP对照组相比，AAV - GAP43 - GFP并未提高RGC的存活率，但AAV - CNTF - GFP注射眼（平均RGC数/视网膜：17450±358个标准误）和AAV - BDNF - GFP注射眼（10200±4064个RGCs/视网膜）的RGC存活率增加。与AAV - CNTF - GFP和AAV - BDNF - GFP注射眼中RGC活力增加一致，这些动物在ON挤压近端有许多βIII - 微管蛋白和GFP阳性纤维。然而，仅在AAV - CNTF - GFP组中，在ON远端可见再生的RGC轴突（1135±367个轴突/神经，挤压后0.5毫米处），一些轴突延伸至视交叉。RGC对CNTF具有免疫反应性，定量逆转录 - PCR显示，用AAV - CNTF - GFP转导的视网膜中CNTF mRNA表达大幅增加。将RGC的AAV - CNTF - GFP转导与自体PN - ON移植相结合，可使RGC的存活和再生效果更佳。在PN移植7周后，每只视网膜有27954（±2833）个存活的RGC，约占成年RGC总数的25%。其中，在向PN移植物中注射荧光金后，每只视网膜有13352（±1868）个RGC被逆行标记。总之，AAV介导的CNTF表达可促进受损成年RGC的长期存活和再生，通过将基因治疗与基于细胞的治疗/干预相结合，这些效果可得到显著增强。
