脐血CD34^＋细胞体外扩增脐血巨核祖细胞的研究  

Ex vivo Expansion of Megakaryocyte Progenitors from Human Umbilical Cord Blood CD34^+ Cells

作　　者：何祎 孟恒星 张宇光 侯士芳 李茜 韩俊领 邱录贵 韩忠朝 

机构地区：[1]协和干细胞基因工程有限公司,天津300384 [2]中国医学科学院、北京协和医学院血液学研究所,血液病医院,天津300020

出　　处：《中国实验血液学杂志》2008年第5期1121-1125,共5页Journal of Experimental Hematology

摘　　要：本研究探讨人脐血CD34+细胞体外扩增的巨核祖细胞的生物学特性及其免疫原性变化,为体外扩增脐血巨核祖细胞的临床应用提供实验依据。采用Ficoll-Hapaque分离法分离人脐血单个核细胞,应用免疫磁珠法(MACS)再分离富集CD34+细胞,在含血小板生成素(TPO,50ng/ml)、白介素-11(IL-11,50ng/ml)和肝素(25U/ml)的无血清液体培养体系中培养14天。用流式细胞术检测扩增产物免疫表型(CD34+、CD41a+、CD61+、CD34+CD41a+及CD34+CD61+)、巨核细胞凋亡率及其表面HLAⅠ、Ⅱ类分子的表达,并进行巨核细胞集落形成单位(CFU-Mk)的检测。结果显示:脐血CD34+细胞能够有效地向巨核细胞分化,CD41a+和CD61+细胞比例在培养第14天达到峰值,CD34+CD41+和CD34+CD61+细胞比例在扩增第7天达到最高峰〔分别为(3.41±2.80)%和(1.89±1.43)%〕;CFU-Mk大集落在扩增第7天达到高峰〔(20.66±32.79)倍〕,小集落在扩增第10天达到高峰〔(435.62±482.65)倍〕;在培养7、10和14天时巨核细胞的凋亡率分别为(19.48±9.64)%、(26.87±9.03)%和(52.46±11.74)%,其中培养7天和10天的凋亡率无显著性差异(p>0.05),培养14天的凋亡率显著高于7天和10天(p均<0.05);巨核细胞表面HLAⅠ、Ⅱ类分子的表达随着扩增天数的延长逐渐降低,其中培养0到10天阶段下降明显。结论:采用TPO+IL11+肝素组合,可以有效地扩增脐血巨核祖细胞;培养7天,CFU-Mk大集落扩增倍数、CD34+CD41+和CD34+CD61+细胞比例均达高峰,这是巨核祖细胞体外扩增的较佳培养时间。This study was purposed to investigate the biological characteristics and immunogenicity changes of ex vivo expanded megakaryocyte progenitors from human umbilical cord blood CD34^＋ cells in order to provide experimental basis for clinical application of ex vivo expanded umbilical cord blood megakaryocyte progenitor cells. Mononuclear cells （MNCs） were obtained from umbilical cord blood by Ficoll-Hapaque density gradient separation. CD34 ^＋ cells were enriched by magnetic cell sorting （MACS）. The selected CD34 ^＋ cells were seeded in serum-free medium stimulated with thrombopoietin （ TPO, 50 ng/ml ）, intefleukin 11 （ IL-11, 50 ng/ml ）, and heparin （ 25 U/ml ） for 14 days. The immunophenotyping （CD34 ^＋, CD41 a ^＋, CD61 ^＋, CD34 ^＋ CD41 a ^＋ and CD34 ^＋ CD61 ^＋ cells） of amplificated products, matured megakaryocyte apoptosis, and expression of humam leukocyte antigen （HLA） class Ⅰ and class Ⅱ molecules were measured by fluorescence-activated cell sorter （FACS）. The number of colony-forming units-megakaryocyte （CFU-Mk） was also evaluated by CFU-Mk assay. The results showed that the umbilical cord blood CD34 ^＋ mononuclear cells could be effectively differentiated into megakaryocytes. The peak expression ratios of CD41a^＋ and CD61 ^＋ cells were all at 14th days, while that of CD34 ^＋ CD41 ^＋ and CD34 ^＋ CD61 ^＋ cells were at 7th day [ （ 3.41 ± 2.80） % and （ 1.89 ± 1.43 ） %, respectively]. The expansion times of large and small CFU-Mk reached peak at 7th day （ 20.66 ± 32. 79）and 10th day （435.62 ±482.65）, respectively. The apoptotic rates of megakaryocytes at 7th, 10th, 14th day were （19. 48 ±9. 64）%, （26. 87 ± 9. 03）%, and（52.46 ± 11. 74）%, respectively. The apoptotic rate of megakaryocytes had no significant difference in 7 and 10 days culture（p 〉0.05）, while that significantly increased in culture for 14 day culture, compared with culture for 7 and 10 days（p 〈0.05, respectively）. The expression

关 键 词：巨核祖细胞 细胞扩增 脐血 细胞凋亡 人类白细胞抗原 CD34^+ 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R331.1[医药卫生—基础医学]