Survival of transplanted neurotrophin-3 expressing human neural stem cells and motor function in a rat model of spinal cord injury

<正>BACKGROUND:Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation,Schwann cell transplantation,olfactory ensheathing cell transplantation,and embryonic neural tissue transplantation.However,there is a need for improved outcomes. OBJECTIVE:To investigate the repair feasibility for rat spinal cord injury using human neural stem cells(hNSCs) genetically modified by lentivirus to express neurotrophin-3. DESIGN,TIME AND SETTING:In vitro cell biological experiment and in vivo randomized,controlled, genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People's Hospital of Yibin,China from March 2006 to December 2007. MATERIALS:A total of 64 adult,female,Wistar rats were used for the in vivo study.Of them,48 rats were used to establish models of spinal cord hemisection,and were subsequently equally and randomly assigned to model,genetically modified hNSC,and normal hNSC groups.The remaining 16 rats served as normal controls. METHODS:hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3.Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension(5×10~5) was injected into the rat spinal cord following T_(10) spinal cord hemisection.A total of 5μL Dulbecco's-modified Eagle's medium was infused into the rat spinal cord in the model group.Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry.Motor function was evaluated using the Basso,Beattie,and Bresnahan(BBB) scale. MAIN OUTCOME MEASURES:The following parameters were measured:expression of neurotrophin-3 produced by genetically modified hNSCs,transgene expression and survival of hNSCs in rats,motor function in rats. RESULTS:hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3.The transplanted hNSCs primarily gathered at,or around,the injection site two weeks following transplantation,and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site.In addition,hNSCs were observed 10 weeks after transplantation.At week 4,BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups,compared with the model group (P<0.05),and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group(P<0.05). CONCLUSION:hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.

BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes. OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3. DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007. MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls. METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale. MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats. RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）. CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.