【摘要】 为了研究hSCGF-α对人脐带间充质干细胞(Human umbilical cord mesenchymal stem cells,hUCMSCs)的作用,采用基因工程技术获得重组人干细胞生长因子-α(Recombinant human Stem Cell Growth Factor-α,rhSCGF-α)。针对SCGF基因的高GC含量,采用PCR两步法获得hSCGF-α基因,插入pET-28a(+)载体质粒,构建重组质粒pET-28a-SCGF-α,转化大肠杆菌BL21(DE3)获得表达菌株。低温20℃诱导24 h,目标重组蛋白人干细胞生长因子-α以可溶性表达为主。通过Ni2+-NTA柱纯化,获得目标重组蛋白,电泳谱带扫描分析蛋白纯度可达90%以上。以巨噬细胞/粒细胞(Granulocyte/macrophage,GM)集落形成实验鉴定重组蛋白的生物学活性,并协同重组人巨噬细胞/粒细胞集落刺激因子(Recombinant human GM-colony stimulating factor,rhGM-CSF)研究其对人脐带间充质干细胞的影响。结果显示,纯化的重组蛋白rhSCGF-α具有生物学活性;hSCGF-α及rhGM-CSF对hUCMSCs均有刺激增殖活性,但协同作用效果最强。更多还原

【Abstract】 To investigate the effect of hSCGF-α on human Umbilical Cord Mesenchymal Stem Cells(hUCMSCs),we obtained hSCGF-α using genetic engineering.hSCGF-α gene was amplified from hUCMSCs cDNA using two-step PCR and was inserted into pET-28a(+) plasmid vector.Induced by IPTG at 20 degrees Celsius for 24 h,the fusion protein expressed in E.coli BL21(DE3) was mainly existing in soluble form.The recombinant hSCGF-α was purified using NI-NTA affinity chromatography and the purity was up to 90%.The colony forming test revealed that combined use hSCGF-α and rmGM-CSF(recombinant murine GM-colony stimulating factor,rmGM-CSF) had granulocyte/macrophage(GM) promoting effects on murine bone marrow GM progenitor.In addition,the results indicated that hSCGF-α and rhGM-CSF had stimulatory effect on hUCMSCs and their synergetic effect was the strongest.更多还原