gas6基因修饰小鼠骨髓间充质干细胞的构建及鉴定

Construction and Identification of gas6 Gene Modified Mouse Mesenchymal Stem Cells

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。

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.