大鼠脊髓损伤后轴突再生与NT-3基因体内转染的影响

Effect of NT-3 gene transfer in vivo on axon regeneration in rats with spinal cord injury

目的:研究阳离子脂质体介导的神经营养因子3基因在大鼠损伤脊髓内的表达,观察外源性神经营养因子3对损伤脊髓轴突再生的作用。方法:试验于2003-10/2004-04在北京军区总医院全军骨科中心实验室进行。采用NYU脊髓打击器制备脊髓完全损伤模型(T9平面)。以直接注射法将脂质体Lipofectamine2000和重组质粒pEGFP-神经营养因子3混合后注入大鼠损伤脊髓。采用反转录聚合酶链反应技术和荧光显微镜检测神经营养因子3基因转染后的体内表达,并通过菜豆凝集素L顺行示踪标记,神经中丝免疫组化染色,神经纤维计数和图像分析来评价神经营养因子3基因转染对轴突再生的影响。结果:经脂质体Lipofectamine2000介导神经营养因子3可有效的转染脊髓组织并得到表达。神经营养因子3转基因后可明显增加神经中丝阳性轴突数目(P<0.01),促进皮质脊髓束再生并通过损伤区。结论:外源性神经营养因子3在损伤区局部大量表达具有神经损伤保护和促进轴突再生作用,表明脂质体介导神经营养因子3体内转染治疗创伤性脊髓损伤的方法是可行的。

AIM:To investigate the expression of cationic liposome mediated neurotrophin 3(NT 3) gene in injured spinal cord of rats,and observe the effect of exogeno us NT 3 on the regeneration of injured axons. METHODS:The experiment was conducted in the Central Laboratory for Orthopaedic s,Orthopadic Center of Chinese PLA,General Hospital of Beijing Military Area Com mand from October 2003 to April.The NYU impactor was used to create the model of complete spinal cord injury(SCI)(T9 plane).The recombinant plasmid pEGFP NT3 a nd Lipofectamine2000 liposome complexes were injected into injured spinal cord d irectly.RT PCR and fluorescence observation confirmed the presence of NT 3 cDN A expression in vivo after NT 3 gene transfer.Using Phaseolus vulgaris leucoagg lutinin(PHA L) anterograde tracing and immunohistochemical staining of neurofil ament,the effect of NT 3 gene transfer in vivo on axon regeneration in the inju red spinal cord was evaluated with nerve fiber counting and imaging analysis. RESULTS:The spinal cord was transfected effectively by injection of the Lipofe ctamine 2000 liposome mediated NT 3 gene transfer and the NT 3 expressed.The number of neurofilament positive axons was significantly increased after NT 3 g ene transfer(P< 0.01),and the regeneration of corticospinal tract in injured reg ion was promoted. CONCLUSION:In vivo transfer of NT 3 cDNA can protect nerve against injury and promote the regeneration of injured axons through increasing local expression o f exogenous NT 3,indicating that liposome mediated NT 3 gene transfer in vivo may be a potential therapy for traumatic SCI.