脐血和骨髓来源的CD34^＋细胞体外扩增巨核祖细胞差异的研究

本研究探讨脐血（CB）和骨髓（BM）来源的CD34^＋细胞体外扩增巨核祖细胞的差异。采用Ficoll—Hypaque分离法分离人CB及BM单个核细胞，免疫磁珠法制备CD34^＋细胞，在含血小板生成素（TPO）、TPO＋白介素11（IL—11）或TPO＋IL11＋肝素的无血清液体培养体系中培养14天。流式细胞术检测扩增产物（CD34^＋、CD41a^＋及CD34^＋CD41a^＋细胞）免疫表型、巨核细胞凋亡率及DNA含量，并以集落形成单位测定法进行粒巨-噬细胞集落形成单位（CFU—GM）、红系爆式集落形成单位（BFU—E）及巨核细胞集落形成单位（CFU—Mk）计数。结果表明：14天培养中，CB来源细胞在总细胞数、CD41a^＋及CD34^＋CD41a^＋细胞扩增倍数上均高于BM（P均〈0．05）。0天CB及BM来源CD34^＋细胞在CFU—GM、BFU—E及总的CFU—Mk的形成能力上无显著性差异（P均〉0．05），但CB来源CD34^＋细胞形成的CFU—Mk以大集落为主，其数量高于BM（P〈0．05）；在培养7、10和14天，CB及BM来源细胞CFU—GM扩增倍数无显著性差异（P均〉0．05），但CB来源细胞的BFU—E及总的CFU—Mk扩增倍数均高于BM（P均〈0．05）。14天培养中CB和BM来源巨核细胞的凋亡率无显著性差异（P均〉0．05）。DNA含量检测发现，14天培养中CB来源巨核细胞始终以2N细胞为主（比例〉90％），而BM来源巨核细胞随着培养时间延长，4N、8N及以上倍体巨核细胞比例逐渐增加。结论：CB来源CD34^＋细胞体外扩增巨核祖细胞能力高于BM，它可能是巨核祖细胞体外扩增较好的来源。

Difierences in Megakaryocyte Progenitor Ex Vivo Expansion between CD34+ Cells Derived from Human Umbilical Cord Blood and Bone Marrow

The purpose of this study was to explore the differences in megakaryocyte progenitor ex vivo expansion between CD34 cells derived from human umbilical cord blood(CB) and bone marrow(BM).Mononuclear cells(MNCs) were obtained from CB or BM by Ficoll-Hypaque density gradient separation.CD34 cells were purified by magnetic cell sorting(MACS).The selected CD34 cells were seeded in serum-free conditions stimulated with thrombopoietin(TPO),TPO interleukin 11(IL-11),or TPO IL11 heparin for 14 days.Amplification product(CD34 ,CD41a ,and CD34 CD41a cells) immunophenotypes,megakaryocyte apoptosis rates and the DNA content were measured by fluorescence-activated cell sorting(FACS).The colony-forming units of granulocytes and monocytes(CFU-GM),burst-forming units of erythrocytes(BFU-E),and colony-forming units of megakaryocytes(CFU-Mk) were also evaluated by the colony-forming units(CFU) assay.The results indicated that CD34 cells derived from CB showed higher expansion ability of total cell counts,CD41a and CD34 CD41a cells than those derived from BM for all days 14 of culture(p<0.05,respectively).There were no significant differences in CFU-GM,BFU-E,and total CFU-Mk counts between CB and BM-derived CD34 cells on day 0(p>0.05,respectively),but CB-derived CFU-Mk seemed mainly large colonies,and the number of large colonies was higher than that from BM(p< 0.05) on day 0.There were no significant differences in expansion ability of CFU-GM between CB and BM-derived cells on days 7,10,and 14 of culture(p>0.05,respectively),but the expansion ability of BFU-E and CFU-Mk derived from CB cells was higher than that from BM(p<0.05,respectively).There were no significant differences in apoptosis rates of megakaryocyte from two source cells for days 14 of culture.Megakaryocytes derived from CB mostly showed the 2N DNA content(>90%) for days 14 of culture,while those cells derived from BM showed the increased DNA content,and 4N,8N or more ploidy cells gradually increased with prolonging of culture time.It is concluded that CB-derived CD34 cells have a greater proliferation potential than that derived from BM,which is therefore proven to be a better cell source for megakaryocyte progenitor expansion in vitro.