The Effect Of Adenovirus-mediated Gene Transfer Of Bcl-2 On Peripheral Nerve Regeneration And Repair

Recently, studies demonstrate that apoptosis has been implicated in the pathogenesis of spinal cord motoneurons death induced by various peripheral nerve injury. The corresponding motoneurons death of spinal cord induced by peripheral nerve injury affects seriously peripheral nerve regeneration and functional recovery. As a result, it has become one of the important directions to study how to protect the neurons closed to death and to reduce or to prevent apoptosis.Replication defective recombinant adenoviral vectors have been demonstrated as an effective method for delivering genes into a variety of cell types and tissues both in vivo and in vitro. It has been proven difficult to transfect genes into neuronal cells because of their lack of cell division. The adenoviral vector's ability to transfer genes into differentiated post-mitotic cells makes them advantageous for a gene delivery system for the nervous system.Bcl-2 gene (B-Lymphoma/Leukemia-2 gene) is a basic member in regulating cell apoptosis and is regarded as an "anti-apoptosis gene" which inhibits apoptosis happening. Recently, Studies demonstrate that Bcl-2 is a major neuroprotective protein in CNS and the increase in Bcl-2 expression level can not only prevent neurons against various injured factors but also promote mammal nervous system neurite regeneration. It is still unknown whether the expression of Bcl-2 in spinal cord antro-horn motoneurons will be increased after transferring Bcl-2 at the site of peripheral nerve damage and whether it will prevent the motoneurons apoptosis induced by peripheral nerveinjury and promote the injured neurite regeneration. After that we use the gene homologous recombinant principle to construct replication defective recombinant adenoviral vectors Ad/s-Bcl-2 and Ad/as-Bcl-2 and Ad/LacZ which mediated Bcl-2 sense cDNA and antisense cDNA and LacZ cDNA transfection respectively. We investigate the possibility of replication-deficient recombinant adenovirus mediated Bcl-2 transfering into the motoneurons for the treatment of peripheral nerve regeneration from in vitro and in vivo. The results are as follows:1. Replication defective recombinant adenoviral vectors Ad/s-Bcl-2 and Ad/as-Bcl-2 and Ad/LacZ have been constructed successfully.2. Ad/s-Bcl-2 and Ad/as-Bcl-2 could infect the culture spinal cord motoneurons and change their Bcl-2 gene expression level. The staining in the neurons transferred with Ad/s-Bcl-2 was significantly deep both in situ hybridyzation and immunohistrochemical stain. The staining in the neurons transferred with Ad/as-Bcl-2 was significantly light. The staining in the neurons transferred with Ad/LacZ has not been different statistically with that in the control.3. Overexpression of Bcl-2 could strongly prevent the apoptosis induced by glutamate and prevent interneuron [Ca2+]i increasing. The numbers of TUNEL positive neurons in group Ad/s-Bcl-2 were significantly fewer than that of in groups Ad/as-Bcl-2 and Ad/LacZ and glutamate and interneuron [Ca2+]i was significantly lower than theirs, but significantly higher than that of control. The numbers of TUNEL positive neurons in group Ad/as-Bcl-2 were significantly higher than that of others and interneuron [Ca2+]i was also higher than theirs. The numbers of TUNEL positive neurons in groups Ad/LacZ and glutamate has not been different statistically.4. Overexpression of Bcl-2 in neurons enhances the survival of cultureneurons. The numbers of neurons phagocytized trypan blue dye in groupAd/s-Bcl-2 in culture day 7 and 9 were significantly fewer than that of other groups. The Ad/LacZ has not been different statistically with control. The numbers of neurons phagocytized trypan blue dye in group Ad/as-Bcl-2 in culture day 7 were significantly higher than that of other groups, and in culture day 9 the all neurons were almost death.5. The remote injection of adenoviral vectors at the site of damage of peripheral nerve injury may result in central nervous system (CNS) gene expression. These studies suggest t...