脐血和外周血来源的巨核细胞体外扩增差异的研究

目的建立外周血(peripheral blood,PB)和脐血(cord blood,CB)来源的CD3+4细胞体外定向诱导扩增巨核细胞(megakaryocyte,MK)的最佳体系,探讨两种来源巨核细胞的扩增差异。方法以Ficoll-Hapaque分离“动员”的外周血和脐血单个核细胞,免疫磁珠分离纯化CD3+4细胞,在含胎牛血清的液体培养体系中,以不同细胞因子组合诱导两种来源的CD3+4细胞,定时进行细胞计数和流式细胞术检测培养体系中CD4+1细胞的含量;同时采用甲基纤维素半固体培养法进行巨核细胞集落培养,测定巨核细胞集落形成单位的数量。结果在血小板生成素(thrombopoietin,TPO)+胎肝酪氨酸激酶配体(FLT-3ligand,FL)+白介素6(interleukin-6,IL-6)+IL-3组合中,外周血来源的CD4+1细胞第10天扩增了131±18倍,脐血来源的CD4+1细胞在培养的第14天扩增了193±25倍,为增殖高峰。均明显高于同来源的其他3组(P<0.05),随着时间的推移两者的CD4+1细胞扩增倍数均呈下降趋势。结论TPO+FL+IL-6+IL-3组合均为CB和PB体外诱导扩增巨核细胞的最佳组合。CB来源的巨核细胞较PB来源的巨核细胞有更强的增生能力,而PB来源的CD3+4细胞产生巨核细胞的时间较CB来源的短,与临床上外周血造血干细胞移植的血小板造血重建快于脐血移植相一致。

Difference in ex vivo expansion of megakaryocytes derived from umbilical cord blood and peripheral blood

OBJECTIVE: Currently, thrombocytopenia is typically observed in patients undergoing hematopioetic stem cell transplantation (HSCT), high-dose chemotherapy or irradiation. Severe thrombocytopenia can cause intestinal and intracranial hemorrhage. To transfuse ex vivo-expanded megakaryocytes (MK) into patients can reinforce the ability of platelet formation and shorten the time of platelet recovery. Therefore it is one of the effective approaches to reduce the danger. The purpose of the present study was to explore the differences in MK expansion between CD(34)(+) stem cells derived from umbilical cord blood (CB) and peripheral blood (PB) and to establish the most optimal culture system. METHODS: Mononuclear cells were isolated by density gradient centrifugation over Ficoll-Hypaque gradient solution. CD(34)(+) cells were isolated by positive selection using an immunomagnetic separation system and the selected CD(34)(+) cells were seeded in Iscove's modified Dulbecco's medium (IMDM) supplemented with fetal calf serum (FCS) and certain kinds of cytokines. After 15 - 17 days of culture, the cells were counted and the content of CD(41)(+) cells was determined by using flow cytometry, and the number of megakaryocyte colony-forming unit (CFU-MK) was simultaneously measured. RESULTS: After the defined days of culture, the cytokine combination of thrombopoietin (TPO) + fetal liver tyrosine kinase ligand (FL) + IL-6 + IL-3 showed to be the most suitable for both PB and CB to obtain high numbers of MK, and to be better than any of the other three groups (P < 0.05). The CD(41)(+) cells from CB were expanded by193 +/- 25 fold on day 14, and those from PB were expanded by 131 +/- 18 fold on day 10. The number of CD(41)(+) cells from both CB and PB decreased. CONCLUSION: For PB and CB, the cytokine combination of TPO + FL + IL-6 + IL-3 is most suitable for obtaining large number of MK and is the best combination for ex vivo MK expansion. MKs from CB seemed to have higher proliferation potential than that from PB, and the optimal culture duration of MK from PB is shorter than that of MK from CB.