胎肝AFT024细胞对脐血CD34^+细胞体外扩增及多药耐药基因转染的影响

Effect of fetal liver AFT024 cells on multidrug resistant gene 1 transfection efficiency and in vitro expansion of CD34^+ cells derived from umbilical cord blood

目的:探讨胎肝AFT024细胞对人类脐血造血干细胞体外扩增的作用及对多药耐药基因(MDR1)转染效率的影响。方法:应用AFT024细胞支持下体外长期培养的方法将人类MDR1基因转入脐血CD34+细胞,观察细胞扩增倍数来检测AFT024细胞对造血干/祖细胞的扩增能力,采用RT-PCR、流式细胞术及耐药集落检测的方法测定基因转染效率、P-糖蛋白(P-gp)的表达及其功能活性。结果:(1)培养21d后AFT024细胞对有核细胞总数(TNC)的扩增没有明显作用,但CD34+细胞和集落形成细胞(CFC)的扩增倍数[(37.9±13.9)倍和(27.1±13.3)倍]均明显高于对照组[(9.1±2.3)倍和(7.7±3.6)倍],差异均有统计学意义(P<0.01)。(2)RT-PCR法可在AFT024组的转染细胞中检测到较高的MDR1mRNA水平,AFT024组基因转染效率(46.0%)明显高于对照组(15.2%);两组P-gp的表达分别为(31.7±10.2)%和(12.6±3.9)%;Rhodamine-123排出试验显示,具有P-gp功能活性的细胞分别为(35.5±11.4)%和(16.6±3.2)%,组间比较差异均有统计学意义(P<0.01)。结论:AFT024细胞具有较强的扩增造血干/祖细胞的能力,并能明显提高MDR1基因在脐血CD34+细胞中的转染效率。

Objective： To investigate the influence of fetal liver AFT024 cells on the transfection efficiency of multi-drug resistant gene 1 （MDR1） and the in vitro expansion of CD34^＋ cells derived from umbilical cord blood. Methods： CD34^＋ cells were isolated from human umbilical cord blood by MACS CD34 Progenitor Cell Isolation Kit and co-cultured with AFT024 cells （AFT024 group） or cultured alone （control group） for 7 days. During the subsequent 14 days, retrovirus carrying MDR1 gene was supplemented twice a week to transfect CD34^＋ cells. On the 7th, 14th and 21st day after culture, the number of total nucleated cells （TNC） was counted, the ratio of CD34^＋ cells was assayed by flow cytometry （FCM） and the number of CD34^＋ cells was calculated, and colony-forming cells （CFC） were counted by methylcellulose cultures. RT-PCR method was used to detect the level of MDR1 mRNA in the transfected cells. The expression and function of P-glycoprotein （P-gp） were evaluated by FCM assay and Rhodamine-123 efflux assay, respectively. The gene transfection efficiency was calculated by drug-resistant colony-forming cells assay. Results： （1） The MDR1 mRNA level in AFT024 group than that in control group. The gene transfection efficiency in AFT024 group was significantly higher than that in control group（46.0% vs 15.2% , P 〈0.01 ））. The expressions of P-gp in AFT024 group and control group were （31.7±10.2） % and （ 12.6±3.9） % , respectively（P 〈 0.01 ）. P-gp efflux functions in AFT024 group and control group were （35.5±11.4） % and （ 16.6±3.2） %, respectively （P 〈 0.01 ）. （2） On the 7th day, the expansion folds of TNCs cells, CD34^＋ cells, and CFCs in control group were slightly higher than those in AFT024 group （ P 〉 0. 05 ）. On the 14th day, the expansion fold of TNCs in control group was significantly higher than that in AFT024 group （P 〈 0.05 ） , while the CD34^＋ cells in the AFT024 group were significantly more than those in control group（ P 〈 0. 05 ）. There was no difference in the expansion folds of CFCs between the 2 groups. On the 21st day, the number of TNCs in AFT024 group was higher than those in control group（ P 〉 0.05 ）. The expansion folds of CD34^＋ cells and CFCs in the AFT024 group were significantly higher than that of the control group（P 〈0.01 ）. Conclusion： AFT024 cells can facilitate MDR1 gene transfection into CD34^＋ cells and improve the expansion of primitive hematopoietic cells in vitro.