shRNA抑制缺氧条件下人视网膜色素上皮细胞低氧诱导因子-1α基因对血管内皮生长因子表达的影响

Down-regulation of vascular endothelial growth factor in human retinal pigment epithelial cells by small hairpin loop RNA targeting hypoxia inducible factor-1α under hypoxia condition

目的探讨缺氧条件下人视网膜色素上皮（hRPE）细胞低氧诱导因子-1α（HIF-1α）对血管内皮生长因子（VEGF）表达的影响。方法利用体外转录法合成针对HIF—1α mRNA序列的靶点之一的小发卡环RNA（shRNA），以化学低氧诱导剂CoCl2模拟RPE细胞缺氧环境，对缺氧培养条件下hRPE细胞的HIF-1α进行RNA干扰，通过逆转录聚合酶链反应（RT-PCR）检测HIF-1α和VEGF mRNA的表达，免疫印迹法检测HIF—1α和VEGF蛋白水平，以正常组、缺氧组及阴性转染组作为对照，观察其对HIF-1α基因的沉默效果及对VEGF表达的抑制作用。结果转染HIF-1α mRNA的特异性shRNA后，RT-PCR检测结果显示缺氧条件下hRPE细胞HIF-1α基因沉默效果为77．1％，VEGF mRNA的表达水平下降了27．8％；免疫印迹法检测结果显示HIF—1α和VEGF蛋白水平显著降低。结论针对HIF-1α mRNA的shRNA能有效地使HIF-1α基因沉默，进而抑制缺氧对VEGF的上调作用。（中华眼科杂志．2007。43：1022—1027）

Objective To explore the effect of hypoxia inducible factor-1 α （HIF-1α） gene on the expression of vascular endothelial growth factor （VEGF） in human retinal pigment epithelial（hRPE） cells under hypoxia conditions by using small hairpin loop RNA （shRNA） to silence HIF-1α. Methods CoCl2 （ 150 μmol/L） was used to simulate the hypoxia environment for hRPE cells. After choosing a target site of HIF-1α mRNA, shRNA was designed and synthesized by this target site. hRPE cells were transfected hy this shRNA in vitro. Then, these cells were cultured under hypoxia conditions （150 μmol/L CoCl2 ）. The mRNA expression of HIF-1α and VEGF was measured by semi-quantitative reverse transcription PCR （ RT- PCR）. The protein level of HIF-1α and VEGF was studied by western blot analysis. Results After hRPE cells were transfected by HIF-1α-specific shRNA, RT-PCR showed that the expression of HIF-1α mRNA was inhibited by 77.1%, and western blot analysis showed that the level of HIF-1α protein was significantly decreased in hRPE cells under hypoxia conditions. Moreover, the expression of VEGF mRNA was inhibited by 27.8% and the level of VEGF protein was also significantly decreased in transfected hRPE cells under hypoxia conditions. Conclusions Under hypoxia conditions, HIF-1α-specific shRNA effectively keeps HIF-1α gene silenced, and consequently down-regulates VEGF expression against hypoxia. These results suggest that HIF-1α is one of the most important cytokines for retinal neovascularization. （Chin J Opbthalmol , 2007,43：1022-1027）