论文部分内容阅读
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 a lentivirus harboring growth-specific 6 (gas6) and to construct and identify mouse bone marrow mesenchymal stem cells (MSCs) stably expressing growth arrest-specific protein 6 (GAS6). Methods: MSCs were isolated and cultured by whole bone marrow adherent method. Flow cytometry was used to detect the MSCs marker molecules. Based on the GenBank gas6 gene sequence, primers were designed and synthesized. Using cDNA extracted from m RNA extracted from MSCs as template The gas6 gene fragment was cloned into the lentivirus expression vector to obtain the Lenti-gas6-GFP-zeocin plasmid and sequenced. A three-plasmid packaging system (shuttle plasmid p Lenti-gas6-GZ, plasmid p SPAX2 and p MD2.G) Packaging the lentivirus and validating the expression of the target gene; fourth generation of MSCs were infected by concentrated lentivirus, MSCs stably expressing GAS6 were screened by zeocin, and the positive cells were detected by flow cytometry Rate and expression of surface marker molecules. Results: The lentivirus carrying gas6 gene was constructed and gas6 gene modified MSCs were established. CONCLUSION: The lentiviral vector can mediate gas6 gene overexpression in mouse MSCs without interfering with the biological characteristics of MSCs, which lays the foundation for further study on the therapeutic effect of gas6 gene-modified MSCs.