转hLIF基因逆转录病毒载体饲养层细胞的建立及其对脐血CD34^+造血干/祖细胞的扩增作用

Establishment of Transgenic Cell Strains Expressing Recombinant Retroviral Vector of hLIF Gene and Their Roles in Proliferation of Umbilical Cord Blood CD34^+ HSPC in vitro

目的建立转人白血病抑制因子(hLIF)基因逆转录病毒载体的饲养层细胞,并观察其对CD34+造血干/祖细胞(HSPC)的扩增作用。方法建立转hLIF基因逆转录病毒载体的饲养层细胞,并用RT-PCR法和ELISA法鉴定目的基因的表达;采用免疫磁珠法分离脐带血CD34+HSPC,流式细胞术检测其纯度;将CD34+HSPC与饲养层细胞共培养,流式细胞术检测各组增殖效果。结果建立的转基因饲养层细胞均有绿色荧光,RT-PCR法和ELISA法证实均有目的基因表达,免疫磁珠法分离的CD34+HSPC纯度可达(95.6±2.58)%,与饲养层细胞共培养后CD34+HSPC可扩增8.74倍,表面黏附分子CXCR4和CD54表达量仍较高。结论建立的转hLIF基因饲养层细胞对CD34+HSPC有一定的扩增作用,且延缓其分化。

Objective To establish the transgenic cell strains expressing recombinant retroviral vector of human leukemia inhibitory factor （hLIF） gene which was supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34＋ hematopoietic stem/progenitor cell （HSPC） in vitro. Methods The recombinant retroviral vector of hLIF gene was transfected into human embryo kidney fibroblasts cells 293T ,and the objective gene was detected by RT-PCR and ELISA .The purity of umbilical cord blood CD34＋ HSPC separated by magnetic-activated cell sorting （MACS） was detected by the flow cytometry. After culturing with feeder layer cells for 7 days, the rate of proliferation was detected by flow cytometry. Results The green fluorescence was observed by fluorescence microscope in the transgenic cell strains,and the objective gene was confirmed by RT-PCR and ELISA.The purity of Umbilical cord blood CD34＋ HSPC separated by magnetic-activated cell sorting （MACS） could reach to （95.6±2.58）%. After culturing with feeder layer cells for 7 days , the CD34＋ ceils were 8.74 times of that in the group containing hLIF than that in group without hLIF, the rate of adhension molecules＇ expression on the surface of CD34＋ cells was also higher in the group containing hLIF than that without hLIF. Conclusion Recombinant retroviral vector of hLIF gene as feeder layer cells could provide a better microenvironment for the growth and proliferation of umbilical cord blood CD34＋ HSPC in vitro which contributes to maintain its muhipotency and undifferentiation state.