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目的探讨经人类巨细胞病毒(HCMV)感染的人脐血造血干细胞(HSC)向淋巴系造血祖细胞(CFU-TL)增殖分化过程中同源盒(hox)b4、b6基因的表达。方法取健康母亲健康足月顺产儿断脐后的胎盘段脐血,采用造血祖细胞体外培养技术,分组以HCMV-AD169病毒液和(或)全反式维A酸(ATRA)持续干预,观察其不同时间点各组细胞集落生成情况,并在鉴定为CFU-TL集落细胞后不同时间点分别提取各组细胞核糖核酸(RNA)并检测其完整性;将提取到的总RNA反转录为互补脱氧核糖核酸,在不同时间点采用实时荧光定量反转录PCR(FQ-RT-PCR)技术检测各组hoxb4、hoxb6基因的表达。结果1.培养的集落细胞经瑞-姬染色法染色,鉴定为CFU-TL。2.各组所提取的RNA结构基本完整。3.人脐血HSC向CFU-TL增殖分化过程中(体外),各组hoxb4、hoxb6基因均有表达,且随培养时间推移,hoxb4基因表达的相对水平逐渐降低;而hoxb6基因表达的相对水平在培养第7天最高,第12天降低。4.与空白对照组比较,ATRA组的hoxb4、hoxb6基因表达的相对水平显著增高,而HCMV组的hoxb4、hoxb6基因表达的相对水平较低。5.与HCMV组比较,HCMV加ATRA组的hoxb4、hoxb6基因表达的相对水平较高。结论1.hoxb4、hoxb6基因在人脐血HSC向CFU-TL增殖分化过程中(体外)均有表达,提示其均与淋巴系统造血密切相关。2.HCMV能下调CFU-TLhoxb4、hoxb6基因的表达(体外),提示HCMV可能通过调控hoxb4、hoxb6基因表达异常导致造血功能异常。3.ATRA能显著上调正常CFU-TLhoxb4、hoxb6基因的表达。
Objective To investigate the expression of b4 and b6 genes in human umbilical cord blood hematopoietic stem cells (HSC) infected by human cytomegalovirus (HCMV) during the proliferation and differentiation of lymphoid hematopoietic progenitor cells (CFU-TL). Methods Umbilical cord blood from placenta of healthy mothers with full-term cesarean section was collected. The hematopoietic progenitor cells were cultured in vitro. The cells were continuously treated with HCMV-AD169 virus solution and / or all-trans retinoic acid (ATRA) The colony formation of each group was observed at different time points, and the RNA of each group was extracted at different time points after CFU-TL colonies were identified. The total RNA was reverse transcribed into Complementary deoxyribonucleic acid, and the expression of hoxb4 and hoxb6 genes in each group was detected by real-time fluorescence quantitative reverse transcription PCR (FQ-RT-PCR) at different time points. Results 1.Cultured colony cells were stained by Reiki-Ji stain and identified as CFU-TL. 2. Each group extracted RNA structure is basically complete. Human umbilical cord blood HSC to CFU-TL proliferation and differentiation (in vitro), each group of hoxb4, hoxb6 gene expression, and with the passage of time, the relative level of hoxb4 gene expression decreased; the relative level of hoxb6 gene expression The highest on the 7th day of culture and the decrease on the 12th day. Compared with the blank control group, the relative levels of hoxb4 and hoxb6 gene expression in ATRA group were significantly increased, while the relative levels of hoxb4 and hoxb6 gene expression in HCMV group were lower. Compared with HCMV group, the relative levels of hoxb4 and hoxb6 gene expression in HCMV plus ATRA group were higher. Expression of hoxb4 and hoxb6 genes in HSCs of human umbilical cord blood during the proliferation and differentiation of CFU-TL (in vitro), suggesting that they are closely related to lymphoid hematopoiesis. HCMV can down-regulate the expression of CFU-TLhoxb4 and hoxb6 genes (in vitro), suggesting that HCMV may cause hematopoietic dysfunction by regulating the expression of hoxb4 and hoxb6 genes. 3.ATRA can significantly upregulate the expression of normal CFU-TLhoxb4 and hoxb6 genes.