转HOXB4基因人骨髓MSC促进脐血CD34~+细胞体外扩增

目的 研究转HOXB4基因的人骨髓间充质干细胞(MSC)对脐血CD34~+细胞体外扩增的支持作用.方法 用病毒载体质粒转染包装细胞293T,收获病毒上清(VCM)感染MSC后,用潮霉素筛选获得转HOXB4的MSC(MSC-HOX).分别将MSC(对照组)与MSC-HOX细胞(实验组)作为CD34~+细胞体外扩增的饲养层细胞,结合细胞因子Fh3/Flk3配体(FL)、促血小板生成素(TPO)、干细胞因子(SCF)和粒细胞-集落生长因子(G-CSF)体外扩增脐血CD34~+细胞10 d,收集所有脐血细胞,检测细胞总数、CD34~+细胞总数,集落细胞形成单位(CFU)数.结果 生产重组逆转录病毒可有效将HOXB4基因转入靶细胞内并表达.脐血CD34~+细胞经体外扩增10 d后,细胞总数和CFU数两组间差异无统计学意义(P>0.05),脐血CD34~+细胞总数和比例高于对照组(P<0.05).结论 转HOXB4基因的人骨髓MSC在脐血CD34~+细胞体外扩增中,可保持CD34~+细胞未分化状态,有潜在的应用价值.

Human bone marrow mesenchymal stem cells transferred HOXB4 support in vitro amplification of blood CD34~+ cells

Objective To investigate the supportive role of human bone marrow mesenchymal stem cells (MSCs) transferred HOXB4 on the in vitro amplification of cord blood CD34~+ cells.Methods Reeombinant retroviral vectors encoding HOXB4 and retroviral genes were co-transinfected into packaging cells 293-HOX.Virus containing medium (VCM) was collected and subsequently centrifugated.Human bone marrow MSCs were infected by VCM, and then selected by exposure to hygromycin.The hygromycin-resistant or unmanipulated MSCs were used as feeding-layer for the in vitro expansion of cord blood CD34~+ cells in the presence of hematopoietic growth factors Flt3/Flk3 ligand(FL), thrombopoietin (TPO), stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF).Results HOXB4 could be readily transferred and expressed in the target cells.After ten days ex vivo amplification, although there were no significant differences in the increasing folds in total cells and colony forming units(CFU) between two arms, MSC-HOX feeding-layer exhibited significant capability to selectively support the amplification of CD34~+ cells.Conclusion HOXB4 could be readily transferred into human bone marrow MSC, during which MSC-HOX feeding-layer showes the ability of preserving the phenotype of hematopoietic stem/progenitor cells.