经人巨细胞病毒感染人脐血造血干细胞向粒-巨噬系和红系祖细胞增殖过程中HOXB6-mRNA及其基因表达

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.2]HCMV组:HCMV-AD169滴度为105pfu,以0.1mL感染培养体系.3]ATRA组:在培养体系分组中加入12μL ATRA,终浓度为60μmol/L.4]HCMV 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.2]HCMV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).3]ATRA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).4]HCMV 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.3]ATRA能上调HOXB6基因的表达.

HOXB6-mRNA and its gene expression in the differentiation process of human cytomegalovirus-infected hematopoietic stem progenitor cells into granulocyte and erythrocyte progenitor cells

BACKGROUND: Is the inhibition of the hematopoietic stem progenitor cell (HSPC) proliferation and differentiation after human cytomegalovirus (HCMV) infection associated with abnormal expression of infected cell proliferated gene?OBJECTIVE: To observe the HOXB6-mRNA expression in the process of proliferation and differentiation of HCMV-infected HSPC into colony-forming unit granulocyte-macrophage (CFU-GM) and colony-forming unit erythroid (CFU-E).DESIGN: A controlled observation.SETTING: Laboratory for Molecular Biology, Affiliated Hospital of Luzhou Medical College, Lanzhou, Gansu Province, China.MATERIALS: All cord blood (CB) specimens were provided by the Obstetrics Department of Affiliated Hospital of Luzhon Medical College. They were collected from the umbilical vein of normal term neonates delivered spontaneously. All neonate mothers were healthy and HBS-Ag-negative. HCMV-IgM antibody revealed by routine ELUSA and HCMV-DNA checked by PCR were undetectable. Written informed consent for the laboratory measurements was obtained from each neonate mother, and the protocol was approved by the hospital's Ethics Committee. HCMV-AD169 strains were obtained from the Institute of Virology, Chinese Academy of Preventive Medicine. All-trans retinoic acid (ATRA, lot No. 20010126) was provided by Chongqing Huapont Pharm. Co., Ltd., China.METHODS: This study was performed at the Laboratory of Molecular Biology (state-level), Affiliated Hospital of Luzhou Medical College of Luzhou Medical College from April 2006 to April 2007. Cord blood mononuclear cells were separated for HSPC culture. According to different interventions, the study consisted of 4 groups. Control group: no HCMV virus solution was added and equal volume of culture medium was added instead. HCMV group: 105 PFU/mL HCMV-AD169 virus solution was added to the culture system. ATRA group: ATRA was added into the cultivation system at the final concentration of 60 μ mol/L. HCMV+ATRA group: ATRA was added into the HCMV group, and its final concentration was also 60 μ mol/L.MAIN OUTCOME MEASURES: In each group, cells were harvested on days 3,7 and 12. HOXB6 mRNA expression levels in CFU-GM and CFU-E were detected by real-time fluorescent-based quantification PCR.RESULTS: In the control group, both CFU-E and CFU-GM expressed HOXB6-mRNA. The HOXB6 mRNA expression was increased as a function of time. The HOXB6-mRNA expressed by CFU-E reached its peak level on day 12, while that expressed by CFU-GM reached its peak level on day 7. Compared to control group, the expression levels of CFU-E and CFU-GM HOXB6-mRNA genes in normal cord blood were significantly lower in the HCMV group (P＜0.05)and significantly higher in the ATRA group (P＜0.05) at each time point after HCMV infection. Furthermore, the expression levels were significantly higher in the ATRA+HCMV group than in the HCMV group at each time point(P＜0.05-0.01).CONCLUSION: HOXB6-mRNA expression is stable and lasting in the proliferation and differentiation of HSPC into CFU-GM and CFU-E. HCMV could down regulate HOXB6 gene expression, and ATRA could up regulate HOXB6 gene expression.