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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。
OBJECTIVE: To study the transfection efficiency and expression duration of recombinant adeno-associated virus (rAAV) -containing β-galactosidase structural gene LacZ and rAAVLacZ virus after skeletal muscle administration rAAV vector-mediated gene of interest Duchenne muscular dystrophy gene therapy to explore the appropriate route of administration. Methods LacZ, packaging plasmid PXX2 and adenovirus vector PXX6 were co-transfected into 293 cells and the recombinant adeno-associated virus vector-mediated rAAVLacZ was packaged. The rAAVLacZ local gastrocnemius muscle was intramuscularly injected into the C57 / BL6 murine bone Muscle, respectively, after a single injection 2 months, 5 months to take muscle sections X-Gal staining. Transfected expression of LacZ gene in hindlimb skeletal muscle was observed by injecting rAAVLacZ through the femoral artery. Results (1) LacZ gene was positive in C57 / BL6 mice after intramuscular injection of rAAVLacZ at 2 months and 5 months, and no expression was found at 5 months. (2) Extensive transfection of LacZ gene in hindlimb skeletal muscle and vascular smooth muscle of rAAVLacZ and C57 / BL6 mice via femoral artery injection. Conclusions Recombinant adeno-associated virus vector-mediated reporter gene LacZ can be efficiently and consistently expressed in the skeletal muscle of C57 / BL6 mice for more than 5 months. Transfected femoral arteries are promising routes for muscle transfection of the lower extremities. This work is neuromuscular Genetic diseases, especially Duchenne muscular dystrophy gene therapy for further research laid the foundation.