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目的比较干扰p27基因表达对骨髓和脐血来源造血祖细胞增殖和造血潜能的影响,并探讨其相关机制。方法以含p27全长反义cDNA逆转录病毒感染经流式细胞仪分选的人骨髓与磁珠分离获得的人脐血来源的CD34+细胞,在含混合生长因子条件下体外培养不同时间,分别观察细胞生长曲线,检测细胞周期,确定细胞增殖能力;半固体培养计数集落形成,确定其造血能力;Westernblot检测p27与CDK2蛋白表达,明确基因转染效果并探讨p27反义cDNA促进细胞扩增的作用途径。以含p27全长正义cDNA和仅含绿色荧光蛋白(GFP)基因的病毒感染细胞为对照。结果与GFP和p27正义cDNA比较,p27反义cDNA对脐血造血祖细胞生长有明显促进作用(P<0.01),至培养第9天p27反义cDNA、p27正义cDNA和GFP组细胞数分别增加了(197.3±47.7)倍、(12.7±8.1)倍和(41.8±30.6)倍;骨髓造血祖细胞数增加不明显,至培养第9天3组细胞数分别增加了(36.0±22.3)倍、(8.7±6.8)倍和(14.1±10.4)倍。p27反义cDNA主要促进脐血和骨髓造血祖细胞S期增加,p27反义cDNA、p27正义cDNA和GFP组脐血造血祖细胞S期细胞百分比为(17.0±4.8)%,(2.0±0.8)%和(4.1±1.8)%;骨髓造血祖细胞则为(8.4±4.4)%,(1.0±0.7)%和(3.8±1.4)%。p27反义cDNA对骨髓和脐血的集落形成能力促进作用高于GFP组(P<0.0
Objective To compare the effect of interfering with p27 gene expression on proliferation and hematopoietic potential of hematopoietic progenitor cells derived from bone marrow and umbilical cord blood and to explore its mechanism. Methods Human umbilical cord blood-derived CD34 + cells isolated from human bone marrow and magnetic beads sorted by retrovirus containing p27 full-length antisense cDNA were separated and cultured in vitro with mixed growth factors Cell growth curve was observed, cell cycle was determined to determine cell proliferation ability; Semi-solid culture count colony formation, to determine its hematopoietic capacity; Western blot detection of p27 and CDK2 protein expression, clear gene transfection and to explore p27 antisense cDNA to promote cell expansion Way of action. The virus-infected cells containing the full-length p27 cDNA and the green fluorescent protein (GFP) gene were used as controls. Results Compared with GFP and p27 sense cDNA, p27 antisense cDNA could significantly promote the growth of cord blood hematopoietic progenitor cells (P <0.01). The number of p27 antisense cDNA, p27 sense cDNA and GFP group increased on the 9th day (197.3 ± 47.7) fold, (12.7 ± 8.1) fold and (41.8 ± 30.6) fold, respectively. The number of hematopoietic progenitor cells in bone marrow did not increase significantly, and the number of cells in group 3 increased by 36.0 ± 22.3 folds, (8.7 ± 6.8) times and (14.1 ± 10.4) times, respectively. The p27 antisense cDNA mainly increased the S phase of cord blood and bone marrow hematopoietic progenitor cells. The percentages of S phase cells in p27 antisense cDNA, p27 cDNA and GFP group were (17.0 ± 4.8)% and (2.0 ± 0.8) % And (4.1 ± 1.8)%, respectively. The bone marrow progenitor cells were (8.4 ± 4.4)%, (1.0 ± 0.7)% and (3.8 ± 1.4)%, respectively. The effect of p27 antisense cDNA on colony-forming ability of bone marrow and cord blood was higher than that of GFP group (P <0.0