重组腺相关病毒介导NgRDN基因促进损伤后视神经轴突再生的实验研究

目的探讨大鼠视网膜神经节细胞（RGC）内转染含NgR^DN的重组腺相关病毒（AAV）后对视神经损伤后再生的影响,并观察其与晶状体损伤及巨噬细胞激活之间的关系。方法将45只Wistar大鼠随机分为3组,A组为玻璃体注射携带绿色荧光蛋白（EGFP）的AAV—EGFP组,B组为玻璃体注射AAV—NgR—EGFP组,C组为玻璃体注射AAV—NgR^DN-EGFP组,3种病毒滴度均为5×10^11v．g/ml。每组又分3个亚组,各亚组5只大鼠,1组为玻璃体内注射rAAV组,2组为玻璃体内注射rAAV＋晶状体损伤组,3组为玻璃体内注射rAAV＋酵母多糖组。于玻璃体内注射rAAV后3周进行视神经夹伤,并于夹伤后4d取右眼视网膜进行植片培养,通过βⅢ微管蛋白染色检测视网膜植块边缘的轴突生长情况;于夹伤后2周进行视神经生长相关蛋白（GAP）-43染色,观察视神经轴突再生情况。结果视网膜植片βⅢ-微管蛋白染色表明,在无髓磷脂培养条件下,AAV—NgR—EGFP、AAV—NgR^DN-EGFP对RGC的轴突再生无影响：而在含髓磷脂培养条件下,即模拟体内RGC的轴突生长环境,B组的轴突再生数量（13个4个）还是长度（36μm±4μm）均低于A组（均P〈0．01）;C1组与A1组比较差异无统计学意义,C2和C3组轴突再生的数量及长度均分别高于A2和A3组,C2组（317个±45个、508μm±44μm）更高于C3组（238个±30个、365μm±48μm,均P〈0．01）;视神经GAP-43染色表明,B组视神经轴突再生明显低于A组,c1组轴突再生与A1组差异无统计学意义．而C2和C3组轴突再生显著高于A2和A3组。结论玻璃体内转染AAV—NgR^DN-EGFP同时使RGC处于激活状态可以促进视神经的轴突再生,NgR^DN可以有效拮抗NgR的作用。

Recombinant adeno-associated virus conducted NgR(DN) enhances axonal regeneration of optic nerve after trauma: experiment with rats

OBJECTIVE: To investigate the effect of recombinant adeno-associated virus conducted NgRDN on the axonal regeneration of optic nerve after trauma. METHODS: Two kinds of adeno-associated virus (AAV), AAV-NgRDN-EGFP containing dominant negative form of Nogo receptor and enhanced green fluorescent protein (EGFP) and rAAV-NgR-EGFP containing Nogo-66 receptor (NgR) and EGFP, were constructed. 45 adult Wistar male rats were randomly divided into three equal groups, all with both eyes as experimental eyes: Groups A, B, and C to undergo injection of rAAV-EGFP, rAAV-NgR-EGFP, and rAAV-NgRDN-EGFP respectively into the vitreous; and each group was subdivided into 3 equal subgroups: subgroups 1 underwent injection of rAAV only, subgroups 2 underwent injection of rAAV and lens trauma, and subgroups 3 underwent injection of rAAV and zymosan. The rats in the Subgroups A2, B2, and C2 underwent. Crush of the optic nerve 2 mm behind the eyeball with optic nerve forceps 3 weeks after the injection. Four days after the crush the right eyes were taken out and the retinal explants were cultured in 2 kinds of culture fluid: with or without myelin. The growth of axons at the edge of retinal explants was observed by immunofluorescent staining with betaIII tubulin. Two weeks after the crush the other eyes were taken out to isolate the optic nerves. Immunofluorescence assay was used to detect the expression of growth associated protein-43 (GAP-43) of optic nerve. The axonal regeneration of optic nerve was observed. RESULTS: betaIII tubulin staining showed that on the condition of culture fluid without myelin both rAAV-NgR-EGFP and rAAV-NgRDN-EGFP showed no effects on the axonal regeneration of retinal ganglion cells (RGCs). However, on the condition of culture fluid with myelin the count of axonal regeneration and the length of regenerated axons of Group B were (13+/-4) and (36 microm+/-4 microm), both significantly lower than those of Group A [(21+/-4) and (83 microm+/-11 microm) respectively, both P<0.01]. There were not significant differences in count of axonal regeneration and length of regenerated axons between Subgroups C1 and A1. The count of axonal regeneration and length of regenerated axons of Subgroups C2 were (317+/-45) and (508 microm+/-44 microm), both significantly higher than those of Subgroup C3 [(238+/-30) and (365 microm+/-48 microm) respectively, both P<0.01], and the values of both Subgroups C2 and C3 were significantly higher than those of Subgroups A2 and A3. The GAP43-positive area in the optic nerve of Group C was significantly larger than that of Group A (P<0.01), and that of Group B was significantly smaller than that of Group A (P<0.01). The GAP43-positive area in the optic nerve of Subgroup A2 was (18.71+/-1.72)x100 microm2, significantly larger than that of Subgroup A3 [(12.75+/-1.02)x100 microm2, P<0.01], and that of Subgroup A3 was significantly larger than that of Subgroup A1 (P<0.01). There were not significant differences in the GAP43-positive area among the subgroups in Group B. CONCLUSION: Transfection of rAAV-NgRDN-EGFP into RGC in an activated status enhances axonal regeneration of optic nerve. NgRDN AAV can inhibit effectively the role of NgR.