间充质干细胞对脐血CD34^＋细胞扩增及其细胞特性变化的影响

本研究探讨脐带间充质干细胞（MSC）对CD34^＋细胞（HSPC）体外扩增的支持作用及对CD34^＋细胞表面标志、归巢黏附分子、集落形成能力等干细胞特征变化的影响。用免疫磁珠法从新鲜分离的脐血单个核细胞分离CD34^＋造血干祖细胞（HSPC）；用MSC饲养层（feeder）制备经^137Cs照射的间充质干细胞饲养细胞（MSC feeder cells）。将CD34^＋细胞接种在不同的培养体系中，实验分为3组：HSPC＋CK组为培养液中加入细胞因子组合（SCF、FL和TPO），HSPC＋MSC组为CD34^＋细胞接种在MSC feeder上，HSPC＋MSC＋CK组同时加入细胞因子组合及MSC饲养细胞。培养后4、7、10、14天计数有核细胞总数（MNC），计算细胞扩增情况；用流式细胞术检测不同处理组间CD34^＋细胞及亚群免疫表型、归巢黏附分子和集落形成能力。结果表明：在2周的培养时间里，3组MNC和CD34^＋细胞均明显增加，MNC扩增数依次HSPC＋MSC＋CK组〉HSPC＋CK组〉HSPC＋MSC组。体外扩增10天内HSPC＋MSC＋CK组MNC得到大量的扩增，同时CD34^＋细胞的扩增亦较高。培养4天3组细胞CD34^＋比例较0天有明显下降（P〈0．01）；扩增后CD34^＋细胞比例：HSPC＋MSC组〉HSPC＋MSC＋CK组〉HSPC＋CK组（P〈0．01）；各组CD34^＋细胞亚型细胞比例有所不同，HSPC＋CK组4天时CD34^＋CD38^-细胞有一过性升高（62．71％），之后迅速降低，7天时为0．05％；HSPC＋MSC组7天时CD34^＋CD38^-细胞比例为18．92％，与HSPC＋CK组比较差异有统计学意义（P〈0．05）。从集落形成分析结果看出：MSC、细胞因子混合组扩增后细胞集落形成能力在不同时间点均维持在较高水平。结论：脐血CD34^＋细胞在体外短期培养（〈7天）下，MSC和细胞因子联合应用能同时使CD34^＋细胞得到明显的扩增并维持造血干祖细胞的生物学特征。

Influence of Mesenchymal Stem Cells on UCB CD34+ CelI Expansion and Characteristics

The aim of this study was to investigate the support effects of mesenchymal stem cells （MSCs） on umbilical cord blood （UCB） CD34^＋ cell （HSPC） expansion in vitro and its influence on cell charateristics including the surface marker of CD34^＋ cells, homing adhesion molecules and colong-forming ability. The mononucleated cells （MNCs） were isolated from UCB, then the CD34^＋ cells were isolated from freshly obtained MNCs by immunomagnetic beads, the MSC feeder cells exposed to γ-ray of 137Cs were prepared by MSC feeder. The CD34^＋ cells were inoculated in different culture media. Experiment was divided into 3 groups ： HSPC ＋ CK group in which cytokines were added to medium （SCF, FL and TPO） ; HSPC ＋ MSC group in which CD34^＋ cells were inoculated on MSC feeder; HSPC ＋ MSC ＋ CK group in which cytokines and MSC feeder cells were added to medium. After culture for 4,7,10, 14 days the MNC amount was counted and expansion ability of CD34^＋ cells was evaluated. The immnnotypes of CD34^＋ cells and subsets, homing adherion molecules and colony-forming ability in different groups detected by flow cytometry. The results showed that the amount of MNCs and CD34^＋ cells all obviously increased during culture for 14 days, the expansion levels of MNCs in 3 groups were HSPC ＋ MSC ＋ CK group 〉 HSPC ＋ CK group 〉 HSPC ＋ MSC group in proper order. Within 10 days of expansion in vitro amount of MNCs obtained significant expansion, meantime the expansion of CD34^＋ cells was higher also. The CD34^＋ count in 3 groups at day 4 of culture dicreased significantly as compared with 0 day of culture （p 〈 0.01 ）. The CD34^＋ cells ratios in 3 groups after expansion were HSPC ＋ MSC group 〉 HSPC ＋ MSC ＋ CK group 〉 HSPC ＋ CK group in proper order（p 〈 0.01 ）, while CD34^＋ subset levels in 3 groups were different, the CD34^＋ CD38^- cells in HSPC ＋ CK group at 4 days of culture increased transiently （62.71%）, then quickly decreased, the CD34^＋ CD38 ^- ce