含hNIS报告基因的乏氧调控重组质粒的构建及其功能鉴定

The Construction and Identification of Hypoxia-regulated Recombinant Plasmid with Reporter Gene hNIS

目的构建乏氧应答启动子调控的人钠/碘共同转运体(hNIS)报告基因重组质粒,为实体瘤微环境乏氧的实时监测提供研究基础。方法合成乏氧反应元件(HRE)的核苷酸片段,克隆入pGL3-promoter,构建为pGL3-promoter-5×HRE载体。然后将扩增的hNIS基因插入pGL3-promoter-5×HRE载体中,构建为pGL3-promoter-5×HRE-NIS重组质粒并进行测序验证。以DMSO处理组作为对照,CoCl_2处理HEK293细胞模拟乏氧;将经验证的pShuttle-NIS质粒转染HEK293乏氧细胞,同时将构建的pcDNA3.1-HIF-1α质粒和pShuttle-NIS质粒按照3:1比例转染HEK293细胞,转染空质粒pcDNA3.1和pShuttle-NIS质粒组作为对照。通过qRT-PCR法检测hNIS基因表达的变化。并用高锝酸盐(^(99m)TcO_4^-)处理双质粒共转染的HEK293细胞,洗脱游离^(99m)TcO_4^-后通过γ计数器采集细胞放射性计数,检测所表达NIS蛋白的功能。结果克隆的基因产物与预期一致,序列无碱基的突变。共转染HIF-1α质粒组及转染pShuttle-NIS的CoCl_2处理组,其hNIS mRNA的表达量显著高于转染空质粒及DMSO处理组(均P<0.01)。共转染HIF-1α质粒组细胞的^(99)TcO_4^-摄取率显著高于空质粒对照组(P<0.01)。结论 pGL3-promoter-5×HRE-NIS重组质粒转染后能介导细胞摄取^(99m)TcO_4^-,为微环境细胞乏氧的核素显像提供实验依据。

Objective To construct pShuttle-5×HRE-CMV-NIS recombinant plasmid regulated by hypoxia-responsive element,which can possibly be used to detect the expression of hypoxia induced factor-la（HIF-lα） gene under hypoxia condition.Methods Artificially synthesize the nucleotide sequences of five copies of hypoxia response elements（HREs） were cloned into pGL3-promoter vector to construct pGL3-promoter-5×HRE vector.Human sodium/iodide symporter（hNIS） gene cDNA was amplified from human genome by RT-PCR,and subcloned into pGL3-promoter-5×HRE vector then was sequenced.After treated with CoCl_2 as hypoxia mimic,HEK293 cells were transfected with recombinant plasmid with hNIS gene,while cells treated with DMSO as the control.Meanwhile,pcDNA3.1-HIF-lαand recombinant hNIS gene vectors were transfected into HEK293 cells at the ratio of 3 to 1,while co-transfection with pcDNA3.1 and pShuttle-NIS vectors cells were taken as the control.NIS mRNA expression was analyzed by qRT-PCR while function of NIS protein was tested by ^（99m）TcO_4 -uptake.Results The sequence data of hNIS gene in recombinant plasmid were in accordance with those reported in the literatures. Compared with control groups,HEK293 cells co-transfected with both pShuttle-5 x HRE-CMV-NIS and HIF-1 a gene vectors and CoCl_2-treated after pShuttle-NIS transfecting presented higher mRNA expressions of NIS and ^（99m）TcO_4 uptake（P0.01）.Conclusion HIF-1 a can be bound to and activate pShuttle-5×HRE-CMV-NIS in cells to accumulate radioactive nuclide mTc04~ and this technique is potential for detection of expression and activity of HIF-1 a,the indicator of cell hypoxia.