Xiong Si-qi, Xia Xiao-bo, Xu Hui-zhuo, Li Yan
Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha 410008, China.
Zhonghua Yan Ke Za Zhi. 2007 Nov;43(11):1028-35.
To evaluate suppression efficiency of hypoxia inducible factor-1 alpha (HIF-1 alpha) specific siRNA derived from recombinant plasmid (pSUPER(H1-siHIF-1 alpha)) on both HIF-1 alpha mRNA and protein expression and concomitant downregulation of expression of downstream angiogenic factor vascular endothelial growth factor (VEGF).
Stable pSUPER(H1-siHIF-1 alpha) expression cell lines were constructed by transient transfection of pSUPER(H1-siHIF-1 alpha) eukaryotic expression vector, followed by puromycin selection. Stable expression pSUPER(H1-siHIF-1 alpha) cell line with highest HIF-1 alpha inhibition efficiency determined by reverse transcription-polymerase chain reaction (RT-PCR) was cultured under normoxia (20% O(2)) and hypoxia conditions (1% O(2)) together with control cells. RT-PCR, western blot and ELISA were used to measure inhibition ability of pSUPER(H1-siHIF-1 alpha) on HIF-1 alpha and VEGF expression.
Compared to the control cells, both mRNA and protein level of HIF-1 alpha and VEGF were dramatically decreased by pSUPER(H1-siHIF-1 alpha) under hypoxia conditions. Under sufficient oxygen supply situation, HIF-1 alpha mRNA level was downregulated by pSUPER(H1-siHIF-1 alpha), but pSUPER(H1-siHIF-1 alpha) did not cause suppression of VEGF expression.
pSUPER(H1-siHIF-1 alpha) could decrease HIF-1 alpha expression under both normoxia and hypoxia conditions. VEGF expression was downregulated under hypoxia conditions only. Consequently, pSUPER(H1-siHIF-1 alpha) might be a powerful tool for the inhibition of retinal neovascularization.
评估重组质粒（pSUPER(H1-siHIF-1α)）来源的缺氧诱导因子-1α（HIF-1α）特异性小干扰RNA（siRNA）对HIF-1α mRNA和蛋白表达的抑制效率，以及对下游血管生成因子血管内皮生长因子（VEGF）表达的协同下调作用。
通过瞬时转染pSUPER(H1-siHIF-1α)真核表达载体构建稳定的pSUPER(H1-siHIF-1α)表达细胞系，随后进行嘌呤霉素筛选。通过逆转录-聚合酶链反应（RT-PCR）确定HIF-1α抑制效率最高的稳定表达pSUPER(H1-siHIF-1α)细胞系，与对照细胞一起在常氧（20% O₂）和缺氧条件（1% O₂）下培养。采用RT-PCR、蛋白质印迹法和酶联免疫吸附测定（ELISA）检测pSUPER(H1-siHIF-1α)对HIF-1α和VEGF表达的抑制能力。
与对照细胞相比，在缺氧条件下，pSUPER(H1-siHIF-1α)可显著降低HIF-1α和VEGF的mRNA及蛋白水平。在充足氧气供应情况下，pSUPER(H1-siHIF-1α)可下调HIF-1α mRNA水平，但不会导致VEGF表达受抑制。
pSUPER(H1-siHIF-1α)在常氧和缺氧条件下均可降低HIF-1α表达。仅在缺氧条件下VEGF表达下调。因此，pSUPER(H1-siHIF-1α)可能是抑制视网膜新生血管形成的有力工具。
