音猬因子信号通路在缺氧诱导的人视网膜色素上皮细胞中血管内皮生长因子表达中的作用

Role of Shh signal transduction pathway in vascular endothelial growth factor expression under hypoxia in cultured human retinal pigment epithelial cells

目的观察探讨音猬因子（Shh）信号转导通路在缺氧诱导的人视网膜色素上皮（hRPE）细胞血管内皮生长因子（VEGF）表达中的作用。方法hRPE-19细胞分为正常对照组（Cont组）、缺氧组。缺氧组加入100μmol／L CoCl2再分为缺氧对照组（CoCl2组）、cyclopamine处理组（CYA组）、二甲基亚砜对照组（DMSO组）。CYA组于造成缺氧前lh加入20μmol／L的cyclopamine进行预处理；DMSO组加入1‰DMSO。各组细胞培养4、8、12、24h。采用荧光定量PCR（RT—PCR）检测细胞内Shh、VEGFmRNA的表达；酶联免疫吸附测定试验（ELISA）检测细胞培养上清液中VEGF蛋白含量。结果RT—PCT检测结果显示，缺氧刺激4、8、12、24h，缺氧组细胞内Shh、VEGFmRNA表达逐渐增高，与Cont组细胞内Shh（F=45．260）、VEGFmRNA（F=264．938）表达比较，差异有统计学意义（P=0．001）。CYA组细胞内Shh、VEGFmRNA表达与CoCl2组比较，差异均有统计学意义（P〈0．01）；DMSO组细胞内Shh、VEGFmRNA表达与CoCI。组比较，差异均无统计学意义（P〉0．05）。ELISA检测结果显示，CoCl2组细胞培养上清液中VEGF蛋白含量与Cont组比较，差异有统计学意义（F=3156．676，P=0．001）。cYA组细胞培养上清液中VEGF蛋白含量较CoCl2组明显降低，差异均有统计学意义（P〈0．01）。结论Shh信号转导通路可能参与了缺氧引起的hRPE细胞中VEGF的表达。

Objective To investigate the role of sonic hedgehog （Shh） signal transduction pathway in the expression of vascular endothelial growth factor （VEGF） under hypoxia in cultured human retinal pigment epithelial （hRPE） cells. Methods ARPE-19 were cultured and divided into normal ARPE-19 （Cont） and hypoxia group （100 /xmol/L CoCl2 Cobalt Chloride ＋ARPE-19） ; hypoxia group was further divided into COC12 group, eyclopamine group （CYA） and dimethyl sulfoxide （DMSO） group. 20 μmol/L cyclopamine was added to the CYA group 1 hour before hypoxia, 1%0 DMSO was added into DMSO group at the same time. The hRPE cells were cultured under hypoxia for 4, 8, 12, 24 hours. The expression of Shh and VEGF were determined by Real-time fluorescent quantitate PCR （RT-PCR）. The amount of VEGF in the hRPE conditioned supernatant was measured using enzyme linked immunosorbent assay （ELISA） at 4, 8, 12, 24 hours, respectively. Results RT-PCR tests showed that the level of Shh and VEGF of hRPE was time dependently increased （Shh： F= 45. 260, P=0.001; VEGF： F：264.938, P=0.001）. The level of Shh and VEGF of hRPE in the group treated with eyclopamine was decreased （P〈0.01）. ELISA tests showed that the amount of VEGF in hRPE supernatant was significantly increased in time-dependent manner （F： 3 156. 676, P = 0. 001）, and it was down-regulated by cyclopamine under hypoxia （P〈0.01） . Conclusion Shh signal transduction pathway could play a role in the VEGF expression induced by hypoxia in hRPE cells.