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目的:制备高纯度滴度的病毒载体,介导外源基因Sonic Hedgehog传递至骨组织工程的种子细胞内。方法:包装重组质粒pSNAV2.0-Shh,生产重组病毒rAAV-Shh,并感染小鼠颅骨前成骨细胞MC3T3-E1,流式细胞仪及实时定量PCR检测基因传递效果。结果:生产出高滴度及纯度的重组病毒rAAV-Shh,能有效感染MC3T3-E1,促进胞内成骨相关因子的表达升高。结论:这种重组病毒rAAV-Shh能有效传递外源基因,为骨组织工程中的种子细胞提供基因传递载体。
OBJECTIVE: To prepare high purity titer of viral vectors and to transfer the foreign gene Sonic Hedgehog into the seed cells of bone tissue engineering. Methods: The recombinant plasmid pSNAV2.0-Shh was packaged and the recombinant virus rAAV-Shh was produced. The mouse osteoblast MC3T3-E1 cells were infected with the transfected cells. Flow cytometry and real-time quantitative PCR were used to detect the gene transfer efficiency. Results: The recombinant virus rAAV-Shh with high titer and purity was produced, which could effectively infect MC3T3-E1 and promote the expression of intracellular osteogenesis-related factors. Conclusion: The recombinant virus rAAV-Shh can effectively transfer foreign genes and provide gene delivery vectors for seed cells in bone tissue engineering.