摘要
【目的】研究巨噬细胞移动抑制因子(MIF)对人血管内皮细胞表达血管生成相关基因的诱导作用。【方法】通过亚克隆，构建原核表达质粒pET22b-MIF，并转化人工程菌B121(DE3)。用Ni-亲合柱分离纯化BL21(DE3)中经IPTG诱导表达的重组MIF。用巨噬细胞移动抑制试验鉴定复性的重组MIF的活性。分别用0、30、60、120ng／mL的重组MIF处理人血管内皮细胞12h，通过Real-time定量PCR检测人血管内皮细胞中VEGF_(165)、FGFR_3、MMP9、TGF-α、PDGF-α的mRNA表达。用体外血管生成试验检测重组MIF诱导人血管内皮细胞的成管腔作用。[结果]正确构建了重组质粒pET22b-MIF。经IPTG诱导，在大肠杆菌中以包涵体形式表达出重组MIF。复性的重组MIF对巨噬细胞移动的抑制水平达30％(P＜0.05)。重组MIF能特异地诱导HVECs中VEGF_(165)、FGFR_3、MMP9、TGF-α、PDGF-α表达，并能特异地诱导人血管内皮细胞形成管腔结构。【结论】在大肠杆菌中成功表达出MIF，MIF能特异地诱导人血管内皮细胞中血管生成相关基因的表达。
[Objective] To investigate the potential role of macrophage migration inhibitory factor （MIF） in promoting angiogenic gene expression in human umbilical vascular endothelial cells （HVECs）. [Methods] Using subcloning technique, the prokaryotic MIF expression plasmid pET22b-MIF was constructed. The MIF gene was induced by IPTG to express 6His-tag fusion protein in E. coli BL21 （DE3） and purified by HisTrap affinity columns. The collected recombinant MIF （rMIF） was renatured and the bioactivity of rMIF was tested by macrophage migration inhibition assay （MMI）. The HVECs were incubated with 0, 30, 60, and 120 ng/mL rMIF for 12 h, and VEGF,65, FGFR3, MMP9, TGF-α and PDGF-α mRNA expression were determined by real-time quantitative PCR test. Using in vitro angiogenesis assay, the tube formation of HVECs induced by MIF was tested. [Results] pET22b-MIF was constructed correctly and transformed into E.coli BL21 （DE3）. The rMIF was successfully expressed in E.coli in inclusive bodies. The result of MMI assay showed that the renatured rMIF inhibited the macrophage cells migration at the rate of 30% （P〈 0.05）. The results of real-time quantitative PCR revealed that MIF specifically promoted VEGF,65, FGFR3, MMP9, TGF-α and PDGF-α genes expression in HVECs. The result of in vitro angiogenesis assay showed that MIF specifically induced tube formation of HVECs. [Conclusion] rMIF is successfully expressed in E. coli and rMIF can promote angiogenic factors expression in HUVECs.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2005年第6期617-621,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家自然科学基金(30300421
30271287) 广东省自然科学基金(015015
33189) 广东省科技计划项目(202B601202) 广东省卫生厅科研基金(B2003002)
关键词
巨噬细胞移动抑制因子
分子克隆
人血管内皮细胞
定量PCR
基因表达
macrophage migration inhibitory factor
molecular cloning
quantitative PCR
human vascular endothelial cell
gene expression
