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目的研究重组腺相关病毒载体(rAAV)介导的人抗肌萎缩蛋白(dystrophin)小基因SMCKA3999对Duchenne肌营养不良(DMD)在病理、肌电图、肌力方面的治疗作用。方法将dystrophin小基因SMCKA3999克隆至rAAV并包装成rAAV SMCKA3999病毒,以5×109病毒颗粒多点注射于DMD模型鼠(mdx鼠)腓肠肌,基因治疗4个月后,以免疫荧光双标法检测肌膜dystrophin基因表达,采用Nicolet肌电及诱发电位仪记录mdx鼠肌电图,基因治疗5个月后以肌肉离体灌注电刺激法测定腓肠肌肌力,观察rAAV SMCKA3999对DMD动物模型鼠的治疗作用。结果rAAVSMCKA3999能有效稳定表达,并使肌膜缺失的dystrophin恢复,明显改善DMD肌电图表现,肌力恢复。结论rAAV SMCKA3999对mdx鼠治疗有效,能显著改善其肌肉功能,应用rAAV介导的dystrophin小基因SMCKA3999基因治疗是临床治疗DMD有希望的方法。
Objective To investigate the therapeutic effect of recombinant human adeno-associated virus vector (rAAV) mediated by human dystrophin gene SMCKA3999 on pathological, EMG and muscular strength of Duchenne muscular dystrophy (DMD). Methods The dystrophin minigene SMCKA3999 was cloned into rAAV and packaged into rAAV SMCKA3999 virus. The 5 × 109 virus particles were injected into gastrocnemius muscle of DMD model mice (mdx mice) 4 months after gene therapy. Membrane dystrophin gene expression, Nicolet EMG and evoked potentials were recorded mdx EMG, gene therapy for 5 months after muscle stimulation in vitro measured gastrocnemius muscle strength observed rAAV SMCKA3999 DMD animal model of rats . Results rAAVSMCKA3999 can effectively and stably express and restore the myofunctional dystrophin. It can significantly improve the EMG performance and muscle strength recovery of DMD. Conclusion rAAV SMCKA3999 is effective in mdx mice and can significantly improve its muscle function. The application of rAAV-mediated SMCKA3999 gene therapy of dystrophin gene is a promising method for the clinical treatment of DMD.