直接转染HOXB4基因和转染HOXB4基因的脐带间充质干细胞体外扩增人脐血CD34＋细胞的研究  

Effects of HOXB4-Transfecting Directly and HOXB4-Transfected HUCMSC on in Vitro Expansion of Human Umbilical Cord Blood CD34^+ Cells

作　　者：贺艳霞[1] 赵春亭[1] 王丽[2] 刘竹珍[1] 吴少玲[1] 冯献启[1] 苏湛[1] 隋爱华[3] 刘晓丹[1] 

机构地区：[1]青岛大学医学院附属医院血液科,山东青岛266003 [2]青岛大学中美干细胞与再生医学中心,山东青岛266003 [3]青岛大学医学院附属医院中心实验室,山东青岛266003

出　　处：《中国实验血液学杂志》2013年第6期1578-1584,共7页Journal of Experimental Hematology

基　　金：山东省自然基金项目(编号ZR2009CM056)

摘　　要：本研究探讨直接转染HOXB4基因和转染HOXB4基因的人脐带间充质干细胞体外扩增人脐血CD34+细胞中有核细胞数、CD34+细胞比例与扩增倍数、细胞周期及集落形成能力的异同。将人脐带间充质干细胞(HUCMSC)分为2组,1组利用慢病毒载体将HOXB4基因转染至HUCMSC并建立滋养层(HOXB4-HUCMSC),另1组建立未转染的滋养层(HUCMSC)。应用免疫磁珠分选系统(MACS)分选出人脐血CD34+细胞,在含细胞因子的培养液中培养48 h后分5组,其中对照组2组:A组CD34+细胞(CD34+cells)为空白对照组,B组空病毒转染CD34+细胞(GFP-CD34+cells)为阴性对照组;实验组共3组:直接转染HOXB4基因组(HOXB4-CD34+cells)为C组;HUCMSC滋养层组(HUCMSC+CD34+cells)为D组;HOXB4-HUCMSC滋养层组(HOXB4-HUCMSC+CD34+cells)为E组。于培养第6、10、14 d计数有核细胞数(NC),第10 d比较不同处理条件对CD34+细胞比例、细胞周期与集落形成能力的影响。结果表明,利用慢病毒载体可将HOXB4基因转染至HUCMSC并检测到表达,成功建立了HUCMSC与HOXB4-HUCMSC滋养层。经过14 d体外培养,5组有核细胞均得到显著扩增,比较表明,其效果依次为HOXB4-HUCMSC滋养层组>直接转染HOXB4基因组>HUCMSC滋养层组>2对照组(P<0.05)。在体外扩增第10 d,5组有核细胞CD34+比例均大幅下降,经计算实验组CD34+细胞扩增倍数较第0 d显著增高,经比较显示,CD34+细胞比例与扩增倍数高低依次为直接转HOXB4基因组>HOXB4-HUCMSC滋养层组>HUCMSC滋养层组>两对照组(P<0.05);流式细胞仪分析细胞周期表明,实验组的S+G2/M期比例较对照组高,其中HOXB4-HUCMSC滋养层组比例最高,为41.57%,高于直接转染HOXB4基因的37.87%与HUCMSC的滋养层组的28.65%(P<0.05)。HOXB4-HUCMSC滋养层组与直接转HOXB4基因组的集落形成能力无差别,但均高于HUCMSC滋养层组与对照组。结论:HOXB4-HUCMSC滋养层可显著扩增CD34+细胞并可保持其干细胞活性,与直接转染HOXB4基因的CD34+细胞可�This study was purposed to investigate the difference of nucleated cell （NC） count, CD34 ＋celi ratio and expansion multiple, cell cycle and colong formation capability in in vitro expansed human umbilical cord bood CD34 ＋ cells from HOXB4-transfecting directly and HOXB4-transfected human umbilical cord mesenchymal stem cells （HUCMSC） by means of prepared feeder layers of HUCMSC. The HUCMSC were divided into 2 groups ：first group, in which HOXB4 gene was transfected into HUCMSC by using lentiviral vecfor, and feeder layers were set up; and second group in which feeder layers for HUCMSC of non-transfected HOXB4 gene were set up. The CD34 ＋ cells were separated from HUCB by mageatic activated cell sorting （MACS）. After culture in medium with cytokines for 2 days, CD34 ＋ cellswere divided into 5 groups, including control group and experimental group. The control groups included CD34 ＋ cells as group A （ blank control group） and GFP-CD34 ＋ cells as group B （ negative control group） and experimental groups included HOXB4-CD34 ＋ cells as group C, HUCMSC ＋ CD34 ＋ cells as group D, HOXB4-HUCMSC ＋ CD34 ＋ cells as group E and ceils in all groups were cultured in vitro. The number of nucleated cells were counted at day 6,10,14 of culture and CD34 immunophenotypes, cell cycle and colony forming capability were measured at day 10 of culture in different conditions. The results indicated that HOXB4 gene could be transfected into HUCMSC by lentiviral vector and feeder layers were set up successfully. After culture for 14 days, the nucleated cells in 5 groups could be amplified effectively, and the expansion levels in 5 groups were in order HOXB4-HUCMSC ＋ CD34 ＋ cell group 〉 HOXB4-CD34 ＋ cell group 〉 HUCMSC ＋ CD34 ＋ cell group 〉 control groups （ P 〈 0.05 ）. At day 10 of in vitro expansion the CD34 ＋ cell percentage decreased significantly in all groups, while the number of CD34 ＋ cell increased in experiment groups, which were in order HOXB4-CD34＋ cells group

关 键 词：脐血 脐带间充质干细胞 CD34+细胞 日删基因 体外扩增 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]