CAJ | 학술논문

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、14 d,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HGPF 40 μg/ml、TP050 ng/ml、IL-310 ng/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HGPF 40 μg/ml、TP050 ng/ml、IL-310 ng/ml是体外扩增骨髓源性肝干细胞的可行方案.
목적 탐토체외확증골수원성간간세포적가행방안.방법 채용밀도제도리심분리법종신선골수중획취부함CD117양성세포화CD184양성세포적조분,연후장세포재체외배양확증0、7d、14 d,확증방안채용함10%자체혈청적고당DMEM배양기체계중화무혈청적고당DMEM배양기체계중가입불동농도적촉간세포생장소(HGPF)、촉혈소판생장소(TPO)화백세포개소3(IL-3),최후용류식세포계수법측정확증세포중CD117양성세포화CD184양성세포적수량변화.결과 함10%자체혈청적고당DMEM배양기체계중가입HGPF 40 μg/ml、TP050 ng/ml、IL-310 ng/ml조적확증효과최호,확증7d적CD117양성세포수량화CD184양성세포수량분별증가6.55배화6.20배,확증14d적CD117양성세포수량화CD184양성세포수량분별증가11.62배화20.57배.결론함10%자체혈청적고당DMEM배양기체계중가입HGPF 40 μg/ml、TP050 ng/ml、IL-310 ng/ml시체외확증골수원성간간세포적가행방안.
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 lugh-glucose DMEM with 10% autologous serum supplemented with 40 μg/ml HGPF, 50 ng/ml TPO, and 10 ng/mIIL-3.