人干细胞生长因子-α基因的克隆、表达及其协同rhGM-CSF对人脐带间充质干细胞的增殖作用

Cloning, expression and characterization of gene encoding human stem cell growth factor-α and its synergetic effect with rhGM-CSF on proliferation of human umbilical cord mesenchymal stem cells

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。

关键词:干细胞生长因子，人脐带间充质干细胞，克隆，协同作用，集落

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.

Key words:stem cell growth factor, human umbilical cord mesenchymal stem cells, cloning, synergetic effect, colony