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[目的]构建携带兔脑红蛋白(neuroglobin,Ngb)基因的重组慢病毒,感染兔骨髓间充质干细胞(bonemarrow mesenchymal stem cells,BMSCs),建立稳定表达兔Ngb的BMSCs/Ngb细胞。[方法]构建携带Ngb基因的重组慢病毒载体,在脂质体Lipofectamine 2000作用下转染293 T细胞,Real-time PCR检测慢病毒滴度;以Ngb重组慢病毒感染BMSCs,通过荧光表达法判定感染复数(multiplicities of infection,MOI),实时荧光定量PCR(real-time PCR)、蛋白质印迹(Western blot)和酶联免疫吸附试验(ELISA)判定感染后的BMSCs中Ngb的表达情况。[结果]构建Ngb重组慢病毒载体,经酶切及测序鉴定完全正确,能够转染293 T细胞并表达,滴度为2×108 TU/ml,Ngb重组慢病毒感染BMSCs最佳MOI值为100,且Ngb重组慢病毒感染BMSCs能持续稳定高水平表达Ngb mRNA和蛋白。[结论]成功构建Ngb重组慢病毒,并将Ngb基因稳定感染至BMSCs中,实现Ngb的持续稳定高水平表达,为Ngb基因修饰的BMSCs移植治疗脊髓损伤(spinal cord injury,SCI)的研究提供基础。
[Objective] To construct recombinant lentivirus carrying neuroglobin (Ngb) gene and infect bone marrow mesenchymal stem cells (BMSCs) to establish BMSCs / Ngb cells stably expressing rabbit Ngb. [Method] Recombinant lentiviral vector carrying Ngb gene was constructed and transfected into 293 T cells by Lipofectamine 2000. The lentivirus titer was detected by Real-time PCR. The BMSCs were infected with Ngb recombinant lentivirus, Multiplicities of infection (MOI), real-time PCR, Western blot and enzyme-linked immunosorbent assay (ELISA) were used to determine the expression of Ngb in infected BMSCs. [Results] The constructed recombinant lentiviral vector of Ngb was completely correct by restriction enzyme digestion and sequencing. It could be transfected into 293T cells and expressed at a titer of 2 × 108 TU / ml. The optimal MOI of Ngb recombinant lentivirus infected BMSCs was 100 , And Ngb recombinant lentivirus-infected BMSCs could consistently and highly expressed Ngb mRNA and protein. [Conclusion] The Ngb recombinant lentivirus was successfully constructed and Ngb gene was stably infected into BMSCs to achieve a sustained and stable high level expression of Ngb, so as to provide a basis for the study of Ngb gene modified BMSCs transplantation in the treatment of spinal cord injury (SCI). .