中文摘要：

目的：构建人来源铁调节蛋白1（IRP1）真核表达质粒,检测该质粒在人肝癌细胞（HepG2）中的表达及其对转铁蛋白受体1（TfR1）和二价金属转运体1（DMT1）表达的影响。方法：从HepG2细胞中提取总RNA,通过逆转录聚合酶链反应获得IRP1的cDNA。根据不同的酶切位点分别构建IRP1 4个分段目的基因,依次连接4段基因并将其构建入真核表达载体pcDNA3.1（＋）,测序验证碱基序列。用FuGENEHD将质粒pcDNA3.1（＋）-IRP1瞬时转染至HepG2。以QRT-PCR方法检测转染pcDNA3.1（＋）-IRP1质粒,观察对IRP1以及其调控基因TfR1和DMT1表达的影响。结果：扩增出IRP1全长cDNA,构建真核表达质粒,4个质粒经相应酶双酶切后,分子量分别为1261bp、502bp、600bp、310bp。经检测质粒转染至HepG2细胞后,IRP1表达显著高于转染空载质粒细胞。结果显示与对照组相比,转染真核pcDNA3.1（＋）-IRP1质粒后IRP1表达显著上调约50倍,TfR1表达增加约3倍,DMT1表达增加约1.5倍,差异均有统计学意义（P〈0.05）。结论：成功构建人IRP1基因真核表达质粒pcDNA3.1（＋）-IRP1,并证明其能在HepG2细胞内高表达,从而影响IRP1调控基因的表达。

英文摘要：

Objective ：To construct the eukaryotic expression plasmid of human Iron regulation protein l and study its expression and function in HepG2 cells. Methods：Total RNA was isolated from HepG2 cells and eDNA library was constructed by reverse transcriptional PCR method. The eDNA prepared was inserted into pcDNA3.1 （＋） vector. All se- quences amplified by PCR were confirmed by complete sequencing. After FuGENEHD-mediated transient transfection of HepG2 with peDNA3.1 （＋）-IRP1 plasmid, the expression levels of IRP1 and its regulated genes were determined by QRT- PCR. Results：DNA sequence analysis demonstrated that pcDNA3. I（＋）-IRP1 plasmid was obtained, which express IRP1 in HepG2. The mRNA expressions of TfR1 and DMT1 were up-regulated significantly when peDNA3.1（＋）-IRP1 was transfeet- ed into HepG2 cells. Conclusion：The pcDNA3.1（＋）-IRP1 plasmid has been successfully constructed with efficient expres- sions in HepG2. The mRNA expressions of TfR1 and DMT1 were increased significantly by approximately 3-fold and 1.5- fold compared with the control.