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造血生长因子促进RV介导LacZ NeoR基因在人骨髓造血细胞中转染的机制 |
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奚永志 孔繁华
摘 要 目的：本文探索了不同组合的造血生长因子SCF、IL3及IL6对逆转录病毒(RV)介导的LacZ－NeoR双标志基因转染人骨髓造血细胞转染效率、表达水平的影响及其相关机理。方法：采用RV转染人骨髓非粘附造血细胞(NABMC)及经SCF、IL3及IL6不同组合预激48 h后的NABMC。经荧光素二－β－D－半乳糖呋喃苷脂(FDG)标记的半乳糖苷酶、G418RCFU－GM及PCR／Sourthern-blot检测NeoR和LacZ基因的表达。结果：早期造血生长因子(HGFs)的预激明显改善了RV介导的LacZ－NeoR基因在人骨髓造血细胞中的转染效率与表达水平，SCF＋IL3＋IL6＞SCF＋IL3＞IL3＋IL6＞SCF＋IL6。氚标记脱氧胸苷(3H－TdR)自杀及5－溴脱氧尿苷－碘化丙啶(BrdU－PI)双标流式细胞仪(FCM)检测显示，HGFs预激后人骨髓造血细胞及CFU－GM的S期比例显著提高。结论：SCF＋IL3＋IL6的联合预激可显著改善RV介导的外源基因在人骨髓造血细胞中的转染效率与表达水平，这可能与HGFs预激后明显提高人骨髓造血细胞及CFU－GM的S期比例密切相关。
关键词 人类；血细胞生成；逆转录病毒科；造血细胞生长因子
Mechanisms of hematopoietic growth factors promote retroviral-mediated LacZ-NeoR double marker genes into hematopoietic cells of human bone marrow
XI Yong-Zhi，KONG Fan－Hua
Department of Immunology,Affiliated Hospital for Academy of Medical Sciences,Beijing(100039)
Abstract AIM：To examine prestimulation effects of different combinations of SCF、IL3 and IL6 on transfer efficiency，expression levels and related mechanism of retroviral-mediated LacZ－NeoR double marker genes into hematopoietic cells of human bone marrow.METHODS：Nonadherent hematopoletic cells,either directly or after prestimulation with different combinations of SCF，IL3 and IL6 for 48 hours,was transfected with supernatants from BAG－PA317 amphotropic packaging cell line in the presence of protamine sulfate.Transfer efficiency and expression levels of retroviral-mediated LacZ－NeoR into these target cell were analyzed by FDG staining of FCM for β－galactosidase,PCR／sourthern-blot and G418 selective culture of CFU－GM separately.RESULTS:Compared to control,prestimulation of hematopoietic cells with different combinations of SCF,IL3 and IL6 markedly enhanced the transfer efficency of LacZ－NeoR double marker genes into hematopoietic cells.The most potent combinations of HGFs were SCF＋IL3＋IL6 (48.4%)＞SCF＋IL3(43.2%)＞IL3＋IL6(41.4%)＞SCF＋IL6（40.5％） respectively.Cell-cycle analysis of hematopoietic [1] [2] [3] 下一页
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