腺病毒介导的LacZ基因靶向性导入脊髓及示踪周围神经的动态过程

目的由损伤的周围神经靶向性导入腺病毒介导的LacZ基因(AdLacZ)至脊髓，动态观察病毒裁体逆行输送到脊髓前角运动神经元、脊髓后根神经节(dorsal root ganglia，DRGs)感觉神经元及基因产物顺行标记周围神经的全过程和特点。方法分别将AdLacZ转染大鼠正中神经和胫神经近断端，然后以10-0无创线吻合神经。在转染后9周内的24个不同时间点取出正中神经组的C5～T1脊髓节段、DRGs连同臂丛，胫神经组的L2～L6脊髓节段、DRGs连同骶丛。将脊髓和DRGs的50μm横切片行X—gal染色和免疫组织化学染色，臂丛和骶丛的整个标本分别行X—gal染色。计数阳性脊髓前角运动神经元、DRGs神经元及周围神经轴突数，研究转基因表达在脊髓前角运动神经元、DRGs神经元及正中神经、胫神经的最早时间、高峰时间和持续时间。结果LacZ基因能特异性、高效表达在损伤周围神经的感觉和运动神经元。转基因在脊髓和周围神经的表达严格限于感染神经的同侧。正中神经组标本各部位的转基因表达均早于胫神经组。胫神经组被标记的运动神经元和感觉神经元数均高于正中神经组。表达持续的时间在运动神经元最短，然后是感觉神经元，在周围神经持续时间最长。在同一组内，转基因表达在DRGs神经元最早．然后是运动神经元，最后是周围神经干，而且被标记的感觉神经元数多于运动神经元数。结论由损伤的周围神经导入的AdLacZ不但在靶神经元高效特异性表达，而且能高效顺行标记神经元突起、周围神经直至吻合口远端的再生轴突。这对周围神经损伤的基因治疗和神经示踪研究有实用价值。

MONITORING RETROGRADE ADENOVIRAL TRANSGENE EXPRESSION IN SPINAL CORD AND ANTEROGRADE LABELING OF THE PERIPHERAL NERVES

OBJECTIVE: Targeted adenoviral gene delivery from peripheral nerves was used to integrally analyse the characterization and time course of LacZ gene (AdLacZ) retrograde transfer to spinal cord and transgene product anterograde labeling of peripheral nerve. METHODS: Recombinant replication-defective adenovirus containing AdLacZ was administrated to the cut proximal stumps of median and tibial nerves in Wister rats. Then the transected nerve was repaired with 10-0 nylon sutures. At different time point post-infection the spinal cords of C5 to T1 attached with DRGs and brachial plexuses, or L2 to L6 attached with DRGs and lumbosacral plexuses were removed. The removed spinal cord and DRCs were cut into 50 microm serial coronal sections and processed for X-gal staining and immunohistochemical staining. The whole specimens of brachial or lumbosacral plexuses attaching with their peripheral nerves were processed for X-gal staining. The number of X-gal stained neurons was counted and the initial detected time of retrograde labeling, peak time and persisting period of gene expression in DRG sensory neurons, spinal cord motor neurons and peripheral nerves were studied. RESULTS: The gene transfer was specifically targeted to the particular segments of spinal cord and DRGs, and transgene expression was strictly unilaterally corresponding to the infected nerves. Within the same nerve models, the initial detected time of gene expression was earliest in DRG neurons, then in the motor neurons and latest in peripheral nerves. The persisting duration of beta-gal staining was shortest in motor neurons, then in sensory neurons and longest in peripheral nerves. The initial detected time of beta-gal staining in median nerve models was earlier in median nerve models compared with that in the tibial nerve models. Although the initial detected time and the beginning of peak duration of beta-gal staining were not same, the decreasing time of beta-gal staining in motor and sensory neurons of the two nerve models were started at about the same day 8 post-infection. The labeled neurons were more in tibial nerve models than that in median nerve models. Within the same models, the labeled sensory neurons of DRGs were more than labeled motor neurons of ventral horn. The beta-gal staining was tenser in median nerves than that in tibial nerves. However the persisting time of beta-gal staining was longer in tibial nerve models. CONCLUSION: The strong gene expression in neurons and PNS renders this system particularly attractive for neuroanatomical tracing studies. Furthermore this gene delivery method allowing specific targeting of motor and sensory neurons without damaging the spinal cord might offer potentialities for the gene therapy of peripheral nerve injury.