间充质干细胞对造血干/祖细胞分化为巨核细胞的影响

目的:探讨间充质干细胞(MSC)共培养对体外诱导脐带血单个核细胞来源的造血干/祖细胞生成巨核细胞的影响。方法:分离得到骨髓和脐带2种来源的MSC,并对它们进行表面标志和多向分化能力的鉴定,同时通过实时定量PCR及对RT-PCR产物的电泳分析,对比相同培养代数下2种MSC表达造血因子的情况;用梯度离心法分离得到单个核细胞,通过直接接触或Trans-well分隔的方式分别与MSC共培养,观察细胞增殖情况,并检测巨核系特异性的表面标志和相关基因的表达。结果:骨髓和脐带来源的MSC均分泌对巨核细胞增殖分化有促进作用的造血因子,与造血干/祖细胞直接共培养,对于巨核细胞的增殖有明显的促进作用,分化效果不明显;在非接触共培养的条件下,对巨核细胞的增殖及分化都产生促进作用,且骨髓来源的MSC较脐带来源的MSC效果更加明显。结论:MSC与脐带血造血干/祖细胞非接触培养,对其向巨核分化和增殖的促进作用明显,本实验所用的骨髓来源MSC促分化效果更好。本研究为今后进一步优化巨核系诱导分化体系奠定了基础,并对未来体外大规模制备巨核系祖细胞应用于临床治疗有一定的指导作用。

Effect of Mesenchymal Stem Cells on Megakaryocyte Proliferation and Differentiation

Objective： To investigate whether human mesenchymal stem cell （MSC） co-culture could promote megakaryoeyte（MK） formation from hematopietic stem/progenitor cells（HS/PC）. Methods： We obtained MSC from bone marrow（BM-MSC） or umbilical eord（UC-MSC）, and characterized their surface markers and differentiation capacity. The expression profiles of hematopoietie cytokines by these MSC were identified and compared by semiquantitative and quantitative RT-PCR. Mononuclear cells were obtained from cord blood by density gradient centrifugation. The impact of MSC co-culture on megakaryocyte formation was compared by using two culture systems： the direct contact system and non-contact system, mononuclear cells incubated only with induction media were set as control. Megakaryocyte development was judged by cell proliferation, surface-marker expression and the detection of MK specific genes. Results： BM-MSC and UC-MSC constitutively expressed hematopoietie cytokines that promote megakaryocyte proliferation and differentiation. MSC co-culture promoted the proliferation and survival of HS/PC. Megakaryocyte differentiation was generally better in the non-contact conditions, BM-MSC appeared to be of extra benefits. Conclusion： Our data suggested that MSC co-cuhure could promote HS/PC expansion, and that MK-differentiation was generally greater in the non-contact co-culture with MSC. In this study, BM-MSC appeared to be better than UC-MSC for megakaryocyte induction. Our results laid the foundation for further optimization of megakaryocyte differentiation system. The ex vivo generated megakaryocytic progenitors will also be useful for clinical transfusion.