重组腺相关病毒载体介导的LacZ基因转染骨胳肌途径的研究

目的研究重组腺相关病毒载体(recombinantadeno-associatedvirusvector,rAAV)介导的β-半乳糖苷酶的结构基因LacZ,构建的rAAVLacZ病毒经不同途径给药后骨骼肌的转染效率、表达持续时间,为rAAV载体介导的目的基因对Duchenne肌营养不良的基因治疗探索合适的给药途径。方法采用报告基因LacZ、包装质粒PXX2、腺病毒成份辅助质粒PXX6三质粒共转染293细胞,包装重组腺相关病毒载体介导的rAAVLacZ,将rAAVLacZ局部腓肠肌肌肉注射转染至C57/BL6鼠骨胳肌,分别于单点注射后2个月、5个月取肌肉切片X-Gal染色。采用经股动脉注射rAAVLacZ观察LacZ基因在后肢骨骼肌的转染表达。结果(1)C57/BL6鼠局部肌注rAAVLacZ后,2个月和5个月时,均呈LacZ基因阳性表达,5个月时无表达减低。(2)经股动脉注射rAAVLacZ,C57/BL6小鼠后肢骨骼肌的肌膜及血管壁平滑肌LacZ基因广泛转染。结论重组腺相关病毒载体介导的报告基因LacZ可在C57/BL6鼠骨胳肌中高效持续表达5个月以上,经股动脉转染是有希望的下肢肌肉转染途径,此工作为神经肌肉遗传病,尤其是Duchenne肌营养不良基因治疗的进一步研究奠定了基础。

Study on the recombinant adeno-associated virus vector carrying LacZ gene expression in the skeletal muscle

Objective To look for a gene delivery route to the treatment of Duchenne muscular dystrophy(DMD). Methods The recombinant adeno associated virus vector(rAAV) carrying a LacZ reporter gene was constructed. rAAVLacZ was delivered into the skeletal muscle tissue of C57/BL6 mice by intramuscalar injection. Then an intraarterial delivery route was taken to reveal whether rAAVLacZ could transduce muscle tissue. Results (1)The LacZ gene was efficiently transduced and expressed persisting for 5 months after intramuscalar injection.(2)The membrane of muscle and smooth muscle of vessel was widely transduced by intra arterial delivery rAAVLacZ. Conclusion These data provide the evidence that rAAVLacZ can efficiently transduce muscle for a long period. Improving intraarterial gene delivery will be promising means for rAAV mediated gene therapy for generalized skeletal muscle of DMD.