脐血巨核祖细胞体外诱导扩增的研究  

Study on the induction and differentiation of megakaryocyte progenitor cell derived from umbilical cord blood

作　　者：陈琳[1] 谢小燕[1] 刘大庆[1] 吕洋[1] 岳文[1] 师伟[1] 习佳飞[1] 张秀媛[1] 南雪[1] 王静雪[1] 周军年[1] 李艳华[1] 何丽娟[1] 姚海雷[1] 李思婷[1] 裴雪涛[1] 

机构地区：[1]军事医学科学院野战输血研究所,干细胞与再生医学研究室,北京100850

出　　处：《中华血液学杂志》2014年第3期187-190,共4页Chinese Journal of Hematology

基　　金：国家高技术研究发展计划（2011AA020105）

摘　　要：目的探讨适用于临床的诱导脐血细胞向巨核细胞分化并扩增的方法。方法将脐血用羟乙基淀粉（HEs）沉降红细胞，以Ficoll液密度梯度分离单个核细胞（MNC），并探索不同细胞因子组合以及不同培养基体外诱导其向巨核细胞分化及扩增的效果。结果HES浓度为15gm沉降红细胞效果最好，可获得更多的有核细胞。分别采用116t（IL．11、IL．6、TPO）、st36（SCF、TPO、IL．3、IL．6）、pt36[血小板衍生生长因子（PDGF）、TPO、IL．3、IL．6]、pst36四种不同细胞因子组合的国产无血清培养基体外诱导培养脐血MNC7d，其中st36组（50ng／mlSCF、50ng／mlTPO、20ng／mlIL-3、50ng／mlIL．6）获得的CD41’CD61’细胞数最高，为（6．79±1．97）×10^4。含st36的进口无血清培养基体外培养7d，细胞存活率为（82．85＞0．64）％，优于含相同因子组合的国产无血清培养基[细胞存活率为（60．90±6．93）％]，且获得CD41^＋CD61^＋细胞数为（18．60-]=1．97）×10^4，高于国产无血清培养基。故以含st36的进口无血清培养基作为培养体系进行后续实验。倒置显微镜观察显示，脐血MNC随培养时间的延长，细胞数增多，细胞体积变大，培养7d细胞增殖明显。体外诱导培养10d的脐血MNC经Wright．Giemsa染色可见原巨核细胞、幼巨核细胞、颗粒型巨核细胞等不同阶段的巨核细胞；诱导培养14dCD41^＋CD61^＋细胞率达到（54．27±6.31）％。巨核细胞集落形成单位（cFu．MK）实验表明，随着诱导时间延长，CFU．MK数呈增加趋势。体外诱导14dCFU．MK数为1236．0±32．9，高于未诱导的脐血MNC，差异有统计学意义（P〈0．01）。收集体外诱导培养10d的细胞上清，加入凝血酶，可见聚集的血小板。结论采用HES浓度15g/L沉降红细胞，含st36四种因子组合的进口无血清培养基诱导巨核祖细胞可获得最佳的细胞数和诱导效率，且诱导的细�Objective To build a protocol of separation and induction of megakaryocytes derived from cord blood mononuclear cells. Methods Red blood cells were precipitated by hydroxyethyl starch （HES）. Mononuclear cells were obtained by density gradient centrifugation with Ficoll. The inducing efficiencies of megakaryocytes by using of different cytokine cocktails and culture media were analyzed. Results The best choicefor erythrocyte sedimentation and high efficency of nucleated cells retrieving were obtained by using of 1.5% HES. The isolated cord blood mononuclear cells were cultured with domestic serum-free medium supplemented with ll6t （IL- 11, IL-6, TPO）, st36 （SCF, TPO, IL-3, IL-6）, pt36 （PDGF, TPO, IL-3, IL-6）or pst36 for 7 days. St36 group （50 ng/ml SCF, 50 ng/ml TPO, 20 ng/ml IL-3 and 50 ng/ml IL-6） yielded the most CD41/CD61 positive [ （6.79±1.97）× 10^4]. The cell viability [ （82.85±0.64）% ] of st36 group by using of imported serum-free medium was better than [ （60.90± 6.93 ）% ] that in domestic medium on day 7 after induction, and CD41/CD61 positive cells count [ （ 18.60± 1.97） × 10^4] were more than domestic serum-free medium group. Therefore, we chose imported serum-free medium containing st36 to induce cord blood mononuclear cells. After a prolonged culture, the total cell numbers increased accompanied with an elevated percentage of CD41/CD61 positive cells, which reached （54.27 ＋ 6.31）% on day 14. Wright-Giemsa staining showed that different phase ceils, such as megakaryoblast, promegakaryocyte and granular megakaryocyte, occurred after 10 days＇ culture. Clone forming unit-megakarocytes （CFU-MK） assay showed that the colonies count increased with the prolonged incubation. CFU-MK colonies were [ 1 236.0~32.9] on day 14, which was higher than that in medium without induction （P〈0.01）. Platelets from megakaryocytes showed agglutination function after 10 days＇ culture. Conclusion 1.5% HES was the best solution to precipitate erythrocytes. The combinat

关 键 词：脐血 单个核细胞 巨核祖细胞 诱导分化 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]