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目的探讨脐血造血干细胞(HSC)向粒-单系(CFU-GM)、红系(CFU-E)和淋巴系祖细胞(CFU-TL)增殖分化过程中HOXB6基因的表达情况及全反式维A酸(ATRA)对其的影响。方法1.采用造血干/祖细胞体外培养技术,以ATRA持续干扰人类脐血造血干/祖细胞,观察HSC经人粒细胞-单核细胞集落刺激因子(GM-CSF)、促红细胞生成素(EPO)和植物血凝素(PHA)分别诱导后,在培养第3天、第7天和第12天的CFU-GM、CFU-E及CFU-TL集落生成情况。2.采用实时荧光定量RT-PCR技术(FQ-RT-PCR)检测造血干细胞增殖分化过程中HOXB6基因的表达水平。结果1.人类造血干细胞向CFU-GM、CFU-E和CFU-TL增殖分化过程中,各组细胞HOXB6基因均有表达。2.随时间推移,CFU-GMHOXB6 mRNA在增殖分化的第3天开始表达,第7天表达最强烈,第12天表达明显减弱;CFU-E HOXB6 mRNA表达在第3天、第7天和第12天均持续增强;而在CFU-TL中,HOXB6 mRNA表达在第7天最强烈,第12天表达减弱。3.与对照组比较,ATRA可上调各组HOXB6基因的表达。结论1.在人脐血CFU-GM、CFU-E和CFU-TL的不同增殖分化阶段,HOXB6基因呈现持续稳定的表达,提示HOXB6基因与人类造血干细胞增殖分化过程密切相关。2.HOXB6基因在人类造血干细胞向CFU-GM、CFU-E和CFU-TL的正常增殖分化过程中呈现时间上的规律性。3.低浓度(60μmol.L-1)的ATRA能显著上调HOXB6基因的表达。
Objective To investigate the expression of HOXB6 gene during the proliferation and differentiation of cord blood hematopoietic stem cells (HSC) into CFU-GM, CFU-E and CFU-TL, Effect of retinoic acid (ATRA) on it. Methods 1. Hematopoietic stem / progenitor cells were cultured in vitro and ATRA was used to continuously interfere with human umbilical cord blood hematopoietic stem / progenitor cells. HSCs were transfected with human granulocyte-monocyte colony-stimulating factor (GM-CSF), erythropoietin CFU-GM, CFU-E and CFU-TL colony formation on day 3, day 7 and day 12 after induction by EPO and phytohemagglutinin (PHA), respectively. The expression of HOXB6 gene in hematopoietic stem cell proliferation and differentiation was detected by real-time fluorescence quantitative RT-PCR (FQ-RT-PCR). Results 1.Human hematopoietic stem cells expressed the HOXB6 gene in the proliferation and differentiation of CFU-GM, CFU-E and CFU-TL. Over time, CFU-GMHOXB6 mRNA began to express on the third day of proliferation and differentiation, the expression was the strongest on the seventh day and was significantly decreased on the 12th day. The expression of CFU-E HOXB6 mRNA on day 3, day 7 and 12 days continued to increase; while in CFU-TL, HOXB6 mRNA expression on the 7th day the strongest, weakened on the 12th day. Compared with the control group, ATRA up-regulated HOXB6 gene expression in all groups. HOXB6 gene showed consistent and stable expression at different stages of proliferation and differentiation of human umbilical blood CFU-GM, CFU-E and CFU-TL, suggesting that HOXB6 gene is closely related to the proliferation and differentiation of human hematopoietic stem cells. The gene sequence of HOXB6 showed the regularity in the normal proliferation and differentiation of human hematopoietic stem cells to CFU-GM, CFU-E and CFU-TL. Low concentration (60μmol.L-1) of ATRA can significantly up-regulate HOXB6 gene expression.3.