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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
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Abstract:
To investigate the influence of fetal liver AFT024 cells on the transfection efficiency of multidrug 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 cocultured 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 colonyforming cells (CFC) were counted by methylcellulose cultures. RTPCR method was used to detect the level of MDR1 mRNA in the transfected cells. The expression and function of Pglycoprotein (Pgp) were evaluated by FCM assay and Rhodamine123 efflux assay, respectively. The gene transfection efficiency was calculated by drugresistant colonyforming 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 Pgp in AFTO24 group and control group were (31.7±10.2)% and (12.6±3.9)%, respectively(P＜0.01). Pgp 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＞005). 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＜005). 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.
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Clc Number:
Q255
