重组人体干细胞因子的表达、复性、纯化及其对脐带血干细胞体外扩增作用研究

Expression,refolding,and purification of recombinant human stem cell factor and determination of its effect on ex vivo expansion of cord blood

目的对重组人体干细胞因子(rhSCF)蛋白在脐带血干细胞体外扩增中的作用进行研究。方法通过大肠杆菌表达系统,经IPTG诱导表达获取SCF包涵体,经透析复性、纯化后获得蛋白。从脐带血中提取单核细胞(MNC),分选其中具有CD34+抗原表位的细胞,使用一定浓度的血小板生成素(TPO)、FLT-3配体(FL)及rhSCF组合对脐带血干细胞进行7 d体外扩增以研究rhSCF的作用。结果成功使用IPTG在大肠杆菌BL21诱导表达rhSCF,包涵体经复性及CM-Sepharose纯化获得高纯度的rhSCF。干细胞体外扩增实验表明rhSCF、FL及TPO联用,具有协同刺激CD34+细胞体外扩增的作用。结论成功建立rhSCF原核表达、复性和纯化体系,为造血干细胞体外扩增体系的优化研究奠定了基础,同时为干细胞因子的临床应用提供了有价值的参考。

Objective To study the effect of rhSCF on ex vivo expansion of cord blood.Methods rhSCF was obtained from inclusion bodies following IPTG-induced expression in E.coli.Purified rhSCF was obtained after dialysis and refolding.MNC was extracted from umbilical cord blood（UCB）,and cells with CD34＋ mark were detected.rhSCF was added together with thrombopoietin（TPO） and FLT-3 ligand（FL） in a 7-day ex vivo cord blood expansion assay.Results Production of the rhSCF was readily induced by IPTG in the E.coli expression system.The purification of rhSCF from inclusion bodies after refolding and CM-Sepharose chromatography can be accomplished with high yield.In ex vivo cord blood expansion assays,the addition of rhSCF to assays containing FL and TPO led to further stimulation of the expansion of CD34＋ cells.Conclusion A reproducible and efficient prokaryotic expression system for rhSCF,along with highly effective purification and refolding procedures,was established.Overall,this study provides a solid foundation for further research on the optimization of ex vivo expansion of cord blood.