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Effects Of Adenovirus HuCT-1 Gene Transfer On Spinal Cord Injury In Rats

Posted on:2003-01-20Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z F ZhangFull Text:PDF
GTID:1104360092975315Subject:Surgery
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There are three main types of therapeutic strategies for spinal cord injury (SCI): rescue of injured neurons from secondary injury with prevention of neuronal death; promotion of target-directed axonal regeneration; and neural replacement by transplantation. In experimental study, examples are various cells or tissue transplantation (e.g. fetal CNS tissue, neural stem cells, and Schwann cells), application of neurotrophic factors, and overexpression of growth-associated gene (e.g. GAP-43, c-Jun, and Bcb). All of these strategies are directly or indirectly provided neurotrophic factors, which promote axonal regeneration and rescue neurons for SCI. The survival of central neurons and axonal regeneration depend on multiple neurotropic factors produced by different neuronal targets. Therefore, besides NTs, it is important to local effective and specific factors into the injured spinal cord.Cardiotrophin-1 (CT-1), an IL-6-related cytokine, cause hypertrophy of cardiac myocytes and has pleiotropic effects on various other cell types. CT-1 has potent survival promoting effects on motor neurons in vitro and in vivo. CT-1-treated progressive motor neuronopathy (pmn) mice showed a significantly reduce degeneration of facial motoneuron cytons and phrenic nerve myelinated axons. This study examined whether adenovirus huCT-1 gene transfer had neuroprotective effect, promotion rubrospinal axons regeneration, and recovery of forelimb function after spinal cord injury in adult rats.Main methods and techniques:1. To express human CT-1, the coding region from plasmid pBSSK-huCTl was cloned into plasmid pMD 18-T by PCR and T-A cloning, then cloned intoprokaryotic GST-fusion expression vector pGEX-2T to give pGEX2T-huCTl. After IPTG induced pGEX2T-huCTl expression in E.coli at different temperature and different time, the soluble GST/huCTl was purified by immobilized glutathione columns. The GST-fusion protein was cleaved by thrombin and purified again.2. The huCT-1 gene was cloned into shuttle plasmid pDC316 to give pDC316-huCTl. Recombinant replication-defective adeno virus vector AdCMV-huCTl was rescued in 293 packaging cells by co-transfection and Cre-mediated recombination of both plasmids pDC316-huCTl and pBHGloxdeltal,3Cre containing Ad5 genome with deletions of packaging signal El and E3 regions. The insert gene and its expression were identified by PCR, RT-PCR, and immunohistochemistry. Another control recombinant adeno virus vector AdCMV-eGFP was constructed by same protocol. Recombinant adenovirus vectors were purified by CsCl banding and titrated by plaque forming test.3. Gel foam saturated with AdCMV-huCTl was left into a C3-4 lateral funiculus hemisection cavity that completely interrupted one RST. 1,4, 8 weeks after lesion, RN neurons survival and RST regeneration were demonstrated with retrograde and anterograde tracing techniques, and function recovery was examined forelimb use asymmetries.Main results and conclusions:1. After the pGEX2T-huCTl-DH5 a cells induced by IPTG at 29癈 for 4hr, the highest expression of level of the recombinant protein is about 1/5 of total cell proteins, and the soluble portion is about 2/5 of fusion protein. Purification of soluble portion and thrombin cleaved fusion protein resulted in 85% and 80% purified recombinant GST-fusion protein and huCT-1 protein respectively.2. Recombinant huCT-1 protected 55% motoneurons in spinal cord against sciatic axotomy in vivo in adult rats, indicating recombinant huCT-1 has biological activity in rat neurons.3. We have constructed two recombinant adeno viral vectors: AdCMV-huCTl and AdCMV-eGFP, which expression cassette with MCMV promoter, foreign DNA, and SV40 PolyA was inserted into El of Ad5 genomic DNA with deletions of E3 regions. The positive huCT-1 mRNA and protein were identified in AdCMV-huCTl transfected NIH 3T3 cells. The titer of virus stocks was generally up to 1010 phaque forming units (pfu) per milliliter.4. Following injury t...
Keywords/Search Tags:spinal cord injury, cardiotrophin-1, GST-fusion expression system, adenovirus vector, red nucleus, rubrospinal tract, gene therapy, axon regeneration, recovery of function, anterograde and retrograde tracing
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