HIF-1α质粒转染NIH_3T_3细胞的体外实验

The transfection experiment of HIF1 plasmid mediated by liposome-plasmid in vitro

目的探讨低氧诱导因子1(HIF-1)在NIH3T3细胞的体外表达。方法首先构建真核表达质粒,以脂质体为介导,体外转染细胞,经RT-PCR,Westernblot,ELISA检测基因在mRNA、蛋白质等方面的表达。结果半定量RT-PCR证实在NIH3T3细胞表达外源基因,Westernblot检测到外源HIF-1蛋白质表达,ELISA试验证明表达的基因产物具有生物活性。结论PcDNA3-HIF1真核表达质粒能够有效地在体外表达目的基因,为进一步的动物实验奠定了基础。

Objective To investigate the HIF1 cDNA plasmid'expression in NIH3T3 cell. Methods Construct the eucaryot1e expression plamid .The NIH3T3 cells were grown in DMEM-high Glucose with 10% FBS.The cells were transfected in six-well dishes with lipofectamine in opti-MEM medium. After the transfection , the culture medium was harvested and the total RNA was extracted from the cells.The expression of human HIF1 mRNA and The protein was detected by The reverse-transcriptase polymerase chain reaction(RT-PCR) and Western blot. The VEGF concentration in the culture medium was assayed by use of mouse VEGF specific ELISA kit. Results Human HIF1 mRNA and protein were detected by semiquantitive RT-PCR and Western blot in the transfected cells.The gene product was able to augument VEGF expression. Conclusion The HIF-1 eucaryote expression plasmid can express bioactive gene product in vitro.