【摘要】 目的:制备携带生长停滞特异性基因6(gas6)的慢病毒,并构建和鉴定稳定表达生长停滞特异性蛋白6(GAS6)的小鼠骨髓间充质干细胞(MSCs)。方法:采用全骨髓贴壁法分离培养MSCs,用流式细胞术检测MSCs标志分子;根据Gen Bank中小鼠gas6基因序列设计并合成上下游引物,以MSCs提取的m RNA制备的c DNA为模板扩增gas6基因片段,克隆入慢病毒表达载体,获得Lenti-gas6-GFP-zeocin质粒并测序鉴定;采用三质粒包装系统(穿梭质粒p Lenti-gas6-GZ、包装质粒p SPAX2和p MD2.G)包装慢病毒,并验证其表达目的基因情况;将浓缩的慢病毒离心感染第4代MSCs,用吉欧霉素(zeocin)筛选培养后获得稳定表达GAS6的MSCs,采用流式细胞术检测其阳性率以及表面标志分子表达情况。结果:构建了携带gas6基因的慢病毒,建立了gas6基因修饰的MSCs。结论:慢病毒载体可介导gas6基因在小鼠MSCs中过表达,且不会干扰MSCs的生物特性,为进一步研究gas6基因修饰的MSCs的治疗作用奠定了实验基础。更多还原

【Abstract】 Objective: To prepare the recombinant lentivirus including growth arrest-specific gene 6(gas6), thenestablish and identify gas6 gene modified mouse bone marrow mesenchymal stem cells(MSCs). Methods: TheMSCs were isolated and propagated by the adhesive screening method, and the marks were detected by flow cytom-etry. The primers of gas6 were designed and synthesized according to the Gen Bank, and the gas6 gene was ampli-fied from c DNA transcipted by total m RNA extracted from the MSCs to construct the p Lenti- gas6-GFP-zeocin.The gas6-lentivirus was prepared through three-plasmid packaging system(shuttle plasmid p Lenti-gas6-GZ, packag-ing plasmids p SPAX2 and p MD2.G). The 4th MSCs were infected with the concentrated gas6- lentivirus throughcentrifugation, and the positive transfected cells were screened with zeocin. The positive rate and gas6 overexpress-ing MSCs were determined by flow cytometry. Results: The recombinant lentivirus incorporating gas6 could infecttargeted cells and express gas6 in host cell. The MSCs with stable overexpression of gas6 was established and itsstem cell characteristic was kept. Conclusion: The gas6 gene modified MSCs mediated by lentiviral vector was es-tablished, which would lay the foundation for the further research.更多还原