肝细胞生长因子对c-kit^+Lin^-细胞的增殖作用

Effects of hepatic growth factor on c-kit^+Lin^- cell proliferation in mouse bone marrow

目的探讨在无血清无基质的条件下,肝细胞生长因子(HGF)和因子组合对扩增c-kit+Lin-细胞的影响。方法采用免疫磁珠法分离小鼠骨髓c-kit+Lin-细胞,研究在干细胞因子(SCF),FLt-3配基(FL),促血小板生成素(TPO)和白血病抑制因子(LIF)组合中添加不同浓度的HGF培养7d,检测细胞总数,c-kit+Lin-细胞扩增倍数及细胞凋亡率。结果各因子组均可显著扩增c-kit+Lin-细胞,扩增倍数2￣8倍不等。其中,10ng/mlHGF组扩增c-kit+Lin-细胞效果最为显著,为8.00倍,细胞总数扩增45.43倍,细胞凋亡率为17.42%。但随剂量加大,虽然细胞总数上升,c-kit+Lin-细胞扩增反而下降。40ng/ml组细胞总数扩增80.50倍,c-kit+Lin-细胞扩增效果不明显,仅为2.87倍,细胞凋亡率最低,为5.91%。结论10ng/ml的HGF能协同SCF+FL+TPO+LIF扩增c-kit+Lin-细胞,使细胞凋亡率降低,其表型并不受影响,但过高剂量会诱导其分化。

Objective To determine the optimal cytokine combinations with hepatic growth factor （HGF） that results in the most significant simultaneous in vitro expansion of c-kit^＋Lin^- cells derived from the bone marrow. Methods c-kit^＋Lin^- cells were isolated from mouse bone marrow using a high-gradient magnetic cell sorting system （MACS） and expanded in the presence of stem cell factor （SCF）, FLt-3 ligand （FL）, leukemia inhibitor factor （LIF） thrombopoietin （TPO） and different concentrations of HGF for 7days in a liquid culture system. The total cell number and Annexin-V-positive cell number were counted, and the antigen expressions were studied with fluorescence-activated cell sorting （FACS）. Results In each group, c-kit^＋Lin^- cells were expanded effectively and rapidly by 2 to 8 folds. Addition of 10 ng/ml HGF into SCF＋FL＋LIF＋TPO resulted in the most significant expansion of c-kit^＋Lin^- and total cells by 8.00 and 45.43 folds, respectively, with cell apoptosis rate of 17.42 %. But as the concentration of HGF increased, the c-kit^＋Lin^- cells and the apoptosis rate decreased. Conclusion HGF at10 ng/ml shows optimal synergistic effect with SCF, FL, LIF and TPO in expansion of c-kit^＋Lin^- cells, and excessive HGF may induce cell differentiation.