造血生长因子促进RV介导LacZ-Neo^R基因在人骨髓造血细胞中转染的机制

Mechanisms of hematopoietic growth factors promote retroviral-mediated LacZ-Neo R double marker genes into hematopoietic cells of human bone marrow

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４８ｈ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。

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-Neo R 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-Neo R 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-Neo R 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 cells made by FCM with double staining of bromodexyuridine and propidium idodide demonstrated an highly proporation of hematopoietic cells in S phase after prestimulation with HGFs.-thymidine suicide tests of cultured hematopoietic progenitor cells also indicated increasing percentage of CFU-GM in S phase after prestimulation with HGFs.CONCLUSION: Prestimlation with SCF+IL3+IL6 combination could effectively promote the transfter efficiency of retroviral-mediated gene into human hematopoietic cells,which are associated with increased proporation of hematopoietic cells in S phase.