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目的克隆血小板衍生生长因子A链(platelet-derived growth factor A chain,PDGF-A)基因,以逆转录病毒载体pLXSN为骨架携带PDGF-A基因转染猪骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,bMSCs),为应用PDGF-A基因修饰bMSCs进行创面修复奠定基础。方法采用RT-PCR二步分离法,以人肝癌细胞总RNA为模板,扩增PDGF-A基因的全长cDNA编码序列,构建PDGF-A基因的逆转录病毒载体pLXSN/PDGF-A,并将其导入病毒包装细胞PA317中,经G418筛选并扩增,以病毒液感染NIH3T3细胞,选出抗性克隆,测其病毒滴度,将高滴度病毒液感染bMSCs。采用RT-PCR、Southern blot和ELISA法,分别检测PDGF-A基因转染bMSCs的效率及PDGF-A在bMSCs中的表达水平;光镜、MTT法、成骨与成脂诱导分别检测bMSCs转染PDGF-A基因后的生长状态、增殖情况及干细胞特性。结果经测序验证,克隆到PDGF-A基因的全长cDNA序列与GenBank所报告的该基因序列完全一致。成功构建了PDGF-A链基因的逆转录病毒载体pLXSN/PDGF-A,并实现了PDGF-A基因在bMSCs的稳定表达。bMSCs转染PDGF-A后的生长状态、增殖情况良好,且仍具干细胞特性。结论成功地将PDGF-A基因克隆到pLXSN载体中,并实现了PDGF-A基因在bMSCs中的稳定表达。bMSCs转染PDGF-A后仍具干细胞特性。
OBJECTIVE: To clone the gene of platelet-derived growth factor A chain (PDGF-A) and use the retroviral vector pLXSN as the backbone to carry the PDGF-A gene into bone marrow-derived mesenchymal stem cells stem cells, bMSCs), which laid the foundation for wound healing using PDGF-A gene modified bMSCs. Methods RT-PCR was used to amplify the full-length cDNA sequence of PDGF-A gene from human hepatoma cell total RNA as a template to construct retroviral vector pLXSN / PDGF-A of PDGF-A gene. The recombinant plasmid was introduced into PA317 cell line and was screened by G418 for amplification. NIH3T3 cells were infected with virus solution. The resistant clones were selected and their virus titer was measured. High-titer virus fluid was used to infect bMSCs. The efficiency of transfection of PDGF-A gene into bMSCs and the expression of PDGF-A in bMSCs were detected by RT-PCR, Southern blot and ELISA respectively. Transfection of bMSCs was detected by light microscopy, MTT, osteogenic and adipogenic assay Growth Status, Proliferation and Stem Cell Characteristics of PDGF-A Gene. Results After sequencing, the full-length cDNA sequence of PDGF-A gene cloned was exactly the same as that reported in GenBank. The retroviral vector pLXSN / PDGF-A of PDGF-A gene was successfully constructed and the stable expression of PDGF-A gene in bMSCs was achieved. The growth of bMSCs transfected with PDGF-A showed good proliferation and still had stem cell characteristics. Conclusion PDGF-A gene was successfully cloned into pLXSN vector and stable expression of PDGF-A gene in bMSCs was achieved. BMSCs transfected with PDGF-A still have stem cell characteristics.