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背景:造血祖细胞被人巨细胞病毒(HCMV)感染后增殖和分化能力受到抑制是否与受染细胞增殖的基因异常表达有关联?目的:观察经人类巨细胞病毒感染的人类造血干祖细胞向粒-巨噬系、红系祖细胞增殖过程中HOXB6mRNA表达。设计:观察对比实验。单位:泸州医学院附属医院分子生物学实验室。材料:所有脐血标本由泸州医学院附属医院产科提供,取自正常足月顺产新生儿断脐后的胎盘段脐血。所有新生儿母亲HBSAg阴性,常规ELISA检测抗HCMV-IgM及PCR检测HCMV-DNA均为阴性,且对本实验知情同意,实验经过医院伦理委员会批准。HCMV-AD169株由中国预防医学科学院病毒研究所提供。全反式维甲酸(ATRA)为重庆华邦制药股份有限公司产品,批号为20010126。方法:实验于2006-04/2007-04在泸州医学院附属医院分子生物学实验室完成。分离脐血单个核细胞,进行造血祖细胞培养。根据实验干预方式不同将细胞分为4组:①对照组:与HCMV组等量的IMDM培养液进行细胞培养。②HCMV组:HCMV-AD169滴度为105pfu,以0.1mL感染培养体系。③ATRA组:在培养体系分组中加入12μLATRA,终浓度为60μmol/L。④HCMV+ATRA组:在HCMV感染组中加入ATRA,终浓度同ATRA组。主要观察指标:分别于培养后3,7,12天收获各组细胞,采用实时荧光定量PCR技术分别检测脐血粒-巨噬系祖细胞和脐血红系祖细胞HOXB6 mRNA表达水平。结果:①对照组红系祖细胞和粒-巨噬系祖细胞均表达HOXB6-mRNA,随着时间推移,表达水平增加,其中,脐血红系祖细胞表达HOXB6-mRNA于第12天达高峰,脐血粒-巨噬系祖细胞HOXB6-mRNA于第7天达高峰。②HCMV组脐血粒-巨噬系祖细胞、红系祖细胞感染人巨细胞病毒后各时间点HOXB6基因表达均低于对照组,差异有显著性意义(P<0.05)。③ATRA组各时间点脐血粒-巨噬系祖细胞、红系祖细胞HOXB6-mRNA表达均高于对照组,差异有显著性意义(P<0.05)。④HCMV+ATRA组各时间点红系祖细胞及粒-巨噬系祖细胞的HOXB6-mRNA表达均高于人巨细胞病毒组,差异有显著性意义(P<0.05~0.01)。结论:①人类造血干祖细胞向粒单系和红系祖细胞增殖分化过程中,HOXB6-mRNA呈现持续稳定的表达。②人巨细胞病毒能使HOXB6基因表达下调。③ATRA能上调HOXB6基因的表达。
BACKGROUND: Whether the proliferation and differentiation of hematopoietic progenitor cells infected with human cytomegalovirus (HCMV) is related to the abnormal gene expression of infected cells? Objective: To observe the effect of human cytomegalovirus-infected human hematopoietic stem / progenitor cells Expression of HOXB6 mRNA during the proliferation of granulocyte - macrophage and erythroid progenitor cells. Design: observe the contrast experiment. Unit: Affiliated Hospital of Luzhou Medical College Molecular Biology Laboratory. MATERIALS: All umbilical cord blood samples were obtained from the obstetrics department of Luzhou Medical College Affiliated Hospital, and were obtained from placental cord cord blood after the normal full-term neonatal cut off the umbilical cord. HBSAg negative in all new mothers, anti-HCMV-IgM in routine ELISA and HCMV-DNA in PCR were negative, and the informed consent of the experiment, the experiment approved by the hospital ethics committee. HCMV-AD169 strain by the Chinese Academy of Preventive Medicine virus Institute. All-trans retinoic acid (ATRA) is a product of Chongqing Winbond Pharmaceutical Co., Ltd., batch number is 20010126. METHODS: The experiment was performed at the Molecular Biology Laboratory of Affiliated Hospital of Luzhou Medical College from April 2006 to April 2007. Cord blood mononuclear cells were isolated for hematopoietic progenitor cell culture. The cells were divided into 4 groups according to different experimental interventions: ① Control group: The same amount of IMDM culture medium was used for cell culture with HCMV group. ② HCMV group: HCMV-AD169 titer of 105pfu infected culture system with 0.1mL. ③ ATRA group: add 12μLATRA in the culture system group, the final concentration is 60μmol / L. ④HCMV + ATRA group: ATRA was added to HCMV infection group, and the final concentration was the same as ATRA group. MAIN OUTCOME MEASURES: Groups of cells were harvested at 3, 7, and 12 days after culture, respectively. HOXB6 mRNA expression levels in umbilical cord blood macrophage-derived progenitor cells and umbilical cord blood erythroid progenitor cells were detected by real-time fluorescence quantitative PCR. Results: ① The HOXB6-mRNA was expressed in erythroid progenitor cells and granulocyte-macrophage-derived progenitor cells of control group, with the increase of HOXB6-mRNA expression over time, in which the expression of HOXB6-mRNA reached the peak on the 12th day in erythroid progenitor cells, HOXB6-mRNA of cord blood granulocyte-macrophage-derived progenitor cells peaked on the 7th day. (2) The expression of HOXB6 gene in HCMV group was significantly lower than that in control group (P <0.05) after HCMV infection and human erythroid progenitor cells were infected with cytomegalovirus. (3) The expression of HOXB6-mRNA in umbilical cord blood macrophage-derived macrophage lineage and erythroid progenitor cells in ATRA group were significantly higher than those in control group at each time point (P <0.05). ④ The expression of HOXB6-mRNA in erythroid progenitor cells and granulocyte-macrophage progenitor cells in HCMV + ATRA group was higher than that in human cytomegalovirus group at each time point (P <0.05-0.01). Conclusion: ①The proliferation and differentiation of human hematopoietic stem / progenitor cells into granulocyte-lineage and erythroid lineage progenitor cells show steady and stable expression of HOXB6-mRNA. ② human cytomegalovirus HOXB6 gene can down-regulate gene expression. ③ATRA can up-regulate HOXB6 gene expression.