| It is estimated that the annual incidence of spinal cord injury (SCI) is approximately 50 cases per million population and it is getting up in recent years. SCI primarily affects young adults. The costly expenses and handicap after the injury has been an overwhelming financial and social burden on the families and nations. This signals an urgent need for studies on its basical researcher and clinical treatments, so as to improve repair and recovery after SCI.The treatment of SCI is based on protection against delayed neuronal death. In recent experimental treatment, several strategies, such as neural nutritional factors supply, gene transfer, tissue factor replacement, have been applied to prevent delayed neural injury, to facilitate axon regeneration, and to improve the microenvironment and interfere gene expression in neurons. Moreover, the axon grows to target cells and reconstruct the anatomic connections are the key points of the functional restoration, which is interfered by physical and chemical barrier constructed by glial scar. How to solve this problem has important significance in functional restoration.N-Bak gene was discovered and renamed by Arumae and colleagues in 2001 in their research of Bak gene expression and function in neurons. Endogenetic N-Bak mRNA is expressed exclusively in neurons, and its 22kD transcriptional product only contains BH3 domain. However, different from other Bcl2 family members, N-Bak is antiapoptotic when overexpressed in NGF-deprived neurons, whereas it efficiently kills transfected nonneuronal cells (such as COS7 and HeLa cells). It has been demonstrated by mutation analysis that an intact BH3 domain is sufficient to kill the nonneuronal cells, whereas other domains in addition to the BH3 domain are necessary to protect apoptotic neurons.In this study, we constructed a N-Bak recombined adenovirus and tired to mediate N-Bak gene transport, so as to investigate the role of N-Bak in neural protection against delayed death and rubrospinal regeneration and exercise function restoration after rubrospinal injury. Main methods and techniques1. A pair of primers for N-Bak were designed which contained EcoR I and Hind III specific sites on each end. PCR was performed with pCR3.1-N-Bak as template and N-Bak fragment was achieved and cloned into pDC316-N-Bak. Simultaneously, the reporter gene EGFP was cloned into pDC316-EGFP. The 293 cells was cotransfected with AdCMV-N-Bak and pBHGlox△E1,3Cre, thus AdCMV-N-Bak and AdCMV-EGFP were constructed by Cre/loxp site intracellular homologous recombination. The recombinant adenovirus was identified by RT-PCR, enzyme digestion and immunohistochemical stain, and then was amplified for CsCl gradient purify and plaque forming test to detect the titer of the recombined virus.2. Cortex neurons and astrocytes of rats were cultured, and apoptosis was induced with H2O2 in neurons. AdCMV-N-Bak transfection was performed in each cell type to investigate the function of N-Bak in neurons and nonneuronal cells.3. C3 partial hemisection injury(C3Hx) was executed in rats, which afterwards were divided into injury group, virus control group and N-Bak treatment group. AdCMV-N-Bak was administered to injury site and RN treatment group for 1w to 8w. FG was injected into C2 and C5 retrogradely tracing survival RN neurons of survival and axon regeration, respectively. Asymmetry tests were applied to investigate the forelimb function restoration in injured animal.Main results and conclusions1. The shultter plasmids pDC316-N-Bak and pDC316-EGFP were constructed successfully, and the nucleotide sequences of pDC316-N-Bak were confirmed by sequencing. The 293 cells were cotransfected with pDC316-N-Bak/ pDC316-EGFP and pBHGlox△E1,3Cre, and thus E1/E3 deletion AdCMV-N-Bak and AdCMV-EGFP were constructed by Cre/loxp site intracellular homologous recombination. The recombinant adenovirus titer was up to 1.3×1010-2.5×1011PFU/ml after amplification and CsCl gradient purification, which is qualified for in vivo and in vitro experiments.2. Apoptosis o...
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