摘要
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)
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2007年第11期1022-1027,共6页
Chinese Journal of Ophthalmology
基金
湖北省自然科学基金资助项目(2006ABA157)