人骨髓源性肝干细胞体外扩增方案的探讨

Protocols for cloning human bone marrow-derived hepatic stem cells in vitro

目的探讨体外扩增骨髓源性肝干细胞的可行方案。方法采用密度梯度离心分离法从新鲜骨髓中获取富含CD117阳性细胞和CD184阳性细胞的组分,然后将细胞在体外培养扩增0、7d、14d,扩增方案采用含10%自体血清的高糖DMEM培养基体系中和无血清的高糖DMEM培养基体系中加入不同浓度的促肝细胞生长素(HGPF)、促血小板生长素(TPO)和白细胞介素3(IL-3),最后用流式细胞计数法测定扩增细胞中CD117阳性细胞和CD184阳性细胞的数量变化。结果含10%自体血清的高糖DMEM培养基体系中加入HGPF40μg/ml、TPO50ng/ml、IL-310ng/ml组的扩增效果最好,扩增7d的CD117阳性细胞数量和CD184阳性细胞数量分别增加6.55倍和6.20倍,扩增14d的CD117阳性细胞数量和CD184阳性细胞数量分别增加11.62倍和20.57倍。结论含10%自体血清的高糖DMEM培养基体系中加入HGPF40μg/ml、TPO50ng/ml、IL-310ng/ml是体外扩增骨髓源性肝干细胞的可行方案。

Objective To explore practical protocols for cloning bone marrow-derived hepatic stem cells in vitro.Methods The cell fraction rich in CD117＋ cells and CD184＋ cells was separated from fresh bone marrow by density gradient centrifugation and cultured for 0,7 and 14 days in high-glucose DMEM supplemented with or without 10% autologous serum or in serum-free high-glucose DMEM.All the media were supplemented with different concentrations of hepatocyte growth promoting factors（HGPF）,thrombopoietin（TPO） and interleukin-3（IL-3）.The quantitative changes of CD117＋ cells and CD184＋ cells were measured by flow cytometry.Results The optimal effect for cell cloning was achieved with high-glucose DMEM with 10% autologous serum group supplemented with 40 μg/ml HGPF,50 ng/ml TPO,and 10 ng/ml IL-3.At day 7 of cell culture in this media,the quantity of CD117^＋ cells and CD184^＋ cells increased by 6.55 and 6.20 folds,and by 11.62 and 20.57 folds at day 14,respectively.Conclusion It is practical for cloning bone marrow-derived hepatic stem cells in high-glucose DMEM with 10% autologous serum supplemented with 40 μg/ml HGPF,50 ng/ml TPO,and 10 ng/ml IL-3.