RNA干扰技术体外抑制低氧诱导因子-1α及血管内皮细胞生长因子表达的研究

Study on suppression of hypoxia inducible factor-la and vascular endothelial growth factor expression by siRNA targeting hypoxia inducible factor-1α

目的研究正常氧及低氧环境下，pSUPER^h1-siHIF-1α。真核表达载体对低氧诱导因子-1α（HIF-1α）表达的抑制效果及由此引起的血管内皮细胞生长因子（VEGF）表达的差异。方法构建pSUPER^h1-siHIF-1α真核表达载体，并建立pSUPER^h1-siHIF-1α稳定转染细胞系。采用半定量逆转录聚合酶链反应（RT-PCR）法，筛选HIF-1α mRNA抑制效率最高的细胞系。然后将其与对照细胞在正常氧（20％O2）及低氧环境（1％O2）下培养，采用半定量RT-PCR、免疫印迹法及ELISA技术检测正常氧及低氧环境下pSUPER^h1-siHIF-1α对HIF-1α及VEGF表达的抑制效果。结果低氧环境下，pSUPER^h1-siHIF-1α可明显抑制HIF-1α和VEGFmRNA及蛋白质的表达。而正常氧环境下，pSUPER^h1-siHIF-1α虽可明显下调HIF-1αmRNA表达水平，但对VEGF mRNA和分泌型蛋白表达水平无明显抑制效果。结论低氧环境下，pSUPER^h1-siHIF-1α能显著抑制HIF-1α的表达，且下调其下游基因VEGF的表达。因此，pSUPER^h1-siHIF-1α可能为视网膜新生血管的基因治疗提供新的途径。（中华眼科杂志，2007，43：1028-1035）

Objective To evaluate suppression efficiency of hypoxia inducible factor-1α （HIF-1α） specific siRNA derived from recombinant plasmid （pSUPER^h1-siHIF-1α） on both HIF-1α mRNA and protein expression and concomitant downregulation of expression of downstream anglogenic factor vascular endothelial growth factor （VEGF）. Methods Stable pSUPER^h1-siHIF-1α expression cell lines were constructed by transient transfection of pSUPER^h1-siHIF-1α eukaryotic expression vector, followed by puromycin selection. Stable expression pSUPER^h1-siHIF-1α cell line with highest HIF-1α inhibition efficiency determined by reverse transcription-polymerase chain reaction （ RT-PCR ） was cultured under nomoxia （ 20% 02 ） and hypoxia conditions （1% 02） together with control cells. RT-PCR, western blot and ELISA were used to measure inhibition ability of pSUPER^h1-siHIF-1α on HIF-1α and VEGF expression. Results Compared to the control cells, both mRNA and protein level of HIF-1α and VEGF were dramatically decreased by pSUPER^h1-siHIF-1α under hypoxia conditions. Under sufficient oxygen supply situation, HIF-1α mRNA level was downregulated by pSUPER^h1-siHIF-1α, but pSUPER^h1-siHIF-1α did not cause suppression of VEGF expression. Conclusions pSUPER^h1-siHIF-1α could decrease HIF-1α expression under both nomoxia and hypoxia conditions. VEGF expression was downregulated under hypoxia conditions only. Consequently, pSUPER^h1-siHIF-1α might be a powerful tool for the inhibition of retinal neovascularization. （Chin J Ophthalmol, 2007,43：1028-1035）