CD34^+造血干/祖细胞在转hLIF基因腺病毒载体饲养层细胞中的扩增及其移植SCID小鼠的实验研究

The proliferation of umbilical cord blood CD34^+ HSPC in transgenic cell strains of hLIF and the experimental study of engraftment in SCID mice

目的:建立转hLIF基因腺病毒载体的饲养层细胞,观察对CD34+造血干/祖细胞的扩增作用,并研究移植辐射损伤模型SCID小鼠的效果。方法:建立转hLIF基因腺病毒载体的饲养层细胞,并用RT-PCR法鉴定目的基因;采用免疫磁珠法分离脐带血CD34+造血干/祖细胞,流式细胞术检测纯度;将CD34+造血干/祖细胞与饲养层细胞共培养,流式细胞术检测各组增殖效果,建立辐射损伤模型SCID小鼠,将扩增后的CD34+造血干/祖细胞经CFDASE荧光标记后移植入SCID小鼠体内,通过RT-PCR和观察荧光标记细胞来检测小鼠内的人源细胞。结果:建立的转基因饲养层细胞均有绿色荧光,RT-PCR法证实有目的基因表达,免疫磁珠法分离的CD34+造血干/祖细胞纯度可达(95.6±2.58)%,与饲养层细胞共培养后CD34+造血干/祖细胞可扩增13.2倍,表面粘附分子CXCR4和CD54表达量仍较高,移植入SCID小鼠四周后,仍可见带有荧光标记的人源细胞,RT-PCR证明人源基因Alu的存在。结论:建立的转hLIF基因腺病毒载体饲养层细胞可以有效地扩增CD34+造血干/祖细胞,延缓其分化,并且有较高的移植效率和造血活性。

Objective：To establish the transgenic cell strains expressing recombinant adenovirus vector of hIJF gene which was supposed to be used as feeder layer cells for tile proliferation of umbilical cord blood CD34^＋ HSPC in vitro and study the SCID mice model of HSPC transplantation. Methods：The recombinant adenovirus vector of hLIF gene was transfected into human embryo kidney fibroblasts cells 293A and the human embryo lung fibroblasts cells WI-38 were infected with the recombinant adenovirus, and the objective gene was detected by RT-PCR. The purity of umbilical cord blood CD34^＋ HSPC separated by magnetic-activated cell sorting （MACS） was detected by flow cytometry. After culturing with feeder layer cells for 7 days, the rate of proliferation was detected by flow cytometry. CD34^＋ HSPCs stained by CFDA SE was injected to the sublethally irradiated SCID mice. Humanized gene was detected by RT-PCR and fluorescence microscope. Results： The green fluorescence was observed by fluorescence microscope in the transgenic cell strains, and the objective gene was confirmed by RT-PCR. 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 13.2 times in group containing hLIF than in group without hLIF. The rates of adhensire molecules＇ expression on the surface of CD34^＋ cells were also higher in the group containing hLIF than without hLIF. Four days after transplanted in SCID mice, fluorescentiy-labeled humanized cells could still be observed. The existence of the humanized gene Alu was confirmed by RT-PCR. Conclusion： Recombinant adenovirus vector of hLIF gene as feeder layer cells can effectively enhance proliferation of umbil- ical cord blood CD34^＋ HSPC in vitro and delay their differentiate, what＇s more, it has high transplant efficacy and haematogenesis activity.