胎盘源性间充质干细胞多药耐药基因的转染与鉴定

Transfer and identification of multidrug resistance gene in mesenchymal stem cells derived from human placenta

目的探讨重组逆转录病毒载体介导mdrl基因转染胎盘源性间充质干细胞（P-MSC）以应用于基因治疗的可行性、安全性。方法Percoll密度梯度离心法自人胎盘组织中分离培养间充质干细胞（MSC），利用重复感染方法将含有mdrl基因的逆转录病毒载体导人P-MSC，经逆转录多聚酶链式反应（RT．PCR）检测mdrlmRNA表达，荧光细胞分选法（FACS）检测目的基因表达，同时对mdrl—MSC的干细胞特性进行鉴定及分析。结果转染细胞中显示mdrlmRNA表达；转染P-MSC表达P．gp的阳性细胞百分率为（27．6±5．1）％，明显高于非转染组的（0．4-t-O．1）％，两组比较差异有统计学意义（t=14．291，P〈0．01）；转染MSC在G0／G1期所占比例为95．40％，符合干细胞增殖特点。超微结构揭示其具有典型的低分化干细胞超微结构特点，且在特异诱导条件下具有向成脂、成骨定向分化潜能。结论由重组逆转录病毒介导mdrl基因体外转染胎盘源性MSC可获得高效的P—gP表达，且转染细胞可依然保持其干细胞特性。

Objective To explore the feasibility and safety of mdrl gene transferred into placenta derived mesenchymal stem cells （P-MSCs） by reconstructed retrovirat vector. Methods Human P-MSCs were isolated and expanded by Percoll density gradient and then transduced repeatedly by reconstructed retroviral vector containing mdrl gene. The transfection and expression of mdrl gene was detected by reverse transcription-polymerase chain reaction （RT-PCR） and fluorescence-activated cell sorting （FACS）. Meanwhile, the biological features of mdrl-MSCs were identified and analyzed. Results The expression of mdrlmRNA was found in transfected cells. The expression of P-glycoprotein （P-gp） encoded by mdrl gene was （27. 6 ＋ 5.1 ） % in the transfected P-MSCs cells versus （0. 4 ＋ 0. 1 ） % in the non-transfected P-MSCs cells （ t = 14. 291, P 〈 0. 01 ）. The percent of P-MSCs at quiescent phase （ GO/G1 phase） was around 95.40% and it was in accord with the characterization of stem cells. The mdrl-MSCs exhibited typical uhrastructures of low-differentiated stem cells. Moreover, they still retained the potency of adipogenic and osteogenic differentiation in the presence of appropriate conditioned media. Conclusion A stable expression of P-gp may be obtained by reconstructed retroviral-mediated transfection in vitro. And transfected MSCs retain the characteristics of stem cells.