He Yan-Xia, Zhao Chun-Ting, Wang Li, Liu Zhu-Zhen, Wu Shao-Ling, Feng Xian-Qi, Su Zhan, Sui Ai-Hua, Liu Xiao-Dan
Department of Hematology. The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China.
Department of Hematology. The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China. E-mail:
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2013 Dec;21(6):1578-84. doi: 10.7534/j.issn.1009-2137.2013.06.040.
This study was purposed to investigate the difference of nucleated cell (NC) count, CD34(+) cell ratio and expansion multiple, cell cycle and colony formation capability in in vitro expanded human umbilical cord blood 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 magmatic activated cell sorting(MACS). After culture in medium with cytokines for 2 days, CD34(+) cells were 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 cells 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> HOXB4-HUCMSC+CD34(+) cell group>HUCMSC+CD34(+) cell group>control groups (P < 0.05). The cell cycle detection showed that the percentage of cells in S+G2/M phase in experiment groups were higher than that in control groups (P < 0.05), and percentage of cells in HOXB4-HUCMSC+CD34(+) cells group was higher (41.57%) than that in HOXB4-CD34(+) cells group(37.87%) and HUCMSC+CD34(+) cell group (28.65%) (P < 0.05). There was no statistical difference in the CFU number between HOXB4-HUCMSC+CD34(+) cell group and HOXB4-CD34(+) cell group, which were both higher than that in HUCMSC+CD34(+) cell group and control groups (P < 0.05).It is concluded that the CD34(+) cells cultured on HOXB4-HUCMSC feeder layers can be amplified significantly and kept the characteristics of stem cells, The feeder lager of HOXB4-HUCMSC is relative safe for amplification of CD34(+) cells in vitro, it possesses the potential useful value.
本研究旨在通过制备人脐带来源的间充质干细胞（HUCMSC）饲养层，探讨直接转染HOXB4基因的人脐血CD34(+)细胞与经HOXB4基因转染的人脐带来源的间充质干细胞（HUCMSC）共培养体系中，体外扩增的人脐血CD34(+)细胞的有核细胞（NC）计数、CD34(+)细胞比例、扩增倍数、细胞周期及集落形成能力的差异。将HUCMSC分为2组：第一组，采用慢病毒载体将HOXB4基因转染至HUCMSC，并建立饲养层；第二组，建立未转染HOXB4基因的HUCMSC饲养层。通过磁珠激活细胞分选（MACS）从脐血中分离CD34(+)细胞。在含细胞因子的培养基中培养2天后，将CD34(+)细胞分为5组，包括对照组和实验组。对照组包括A组CD34(+)细胞（空白对照组）和B组GFP-CD34(+)细胞（阴性对照组），实验组包括C组HOXB4-CD34(+)细胞、D组HUCMSC+CD34(+)细胞、E组HOXB4-HUCMSC+CD34(+)细胞，所有组细胞均进行体外培养。在培养的第6、10、14天计数有核细胞数量，并在培养第10天检测不同条件下的CD34免疫表型、细胞周期及集落形成能力。结果表明，慢病毒载体可将HOXB4基因转染至HUCMSC并成功建立饲养层。培养14天后，5组细胞均能有效扩增，扩增水平依次为HOXB4-HUCMSC+CD34(+)细胞组>HOXB4-CD34(+)细胞组>HUCMSC+CD34(+)细胞组>对照组（P<0.05）。体外扩增第10天，所有组CD34(+)细胞百分比均显著下降，而实验组CD34(+)细胞数量增加，依次为HOXB4-CD34(+)细胞组>HOXB4-HUCMSC+CD34(+)细胞组>HUCMSC+CD34(+)细胞组>对照组（P<0.05）。细胞周期检测显示，实验组S+G2/M期细胞百分比高于对照组（P<0.05），且HOXB4-HUCMSC+CD34(+)细胞组细胞百分比（41.57%）高于HOXB4-CD34(+)细胞组（37.87%）和HUCMSC+CD34(+)细胞组（28.65%）（P<0.05）。HOXB4-HUCMSC+CD34(+)细胞组与HOXB4-CD34(+)细胞组的集落形成单位（CFU）数量无统计学差异，二者均高于HUCMSC+CD34(+)细胞组和对照组（P<0.05）。结论：HOXB4-HUCMSC饲养层培养的CD34(+)细胞可显著扩增并保持干细胞特性，HOXB4-HUCMSC饲养层对体外扩增CD34(+)细胞相对安全，具有潜在应用价值。
