目的：观察缺氧状态下人视网膜色素上皮(retinal pigment epithelium， RPE)细胞中盘状结构域受体2(discoidin domain receptor 2， DDR2)的表达，观察Ⅰ型胶原缺氧刺激下RPE细胞中DDR2，缺氧诱导因子-1α(hypoxia inducible factor-1α， HIF-1α)和血管内皮生长因子(vascular endothelial growth factor， VEGF)的表达，探讨Ⅰ型胶原和DDR2在脉络膜新生血管(choroidal neovascularization， CNV)发生中的作用。

方法：对照实验研究。将体外培养的人RPE细胞置于含终浓度为200μmol/L CoCl₂的培养液中培养以建立RPE细胞化学缺氧模型。在缺氧后即刻(即常氧状态下)、2，6，12和24h终止缺氧，用免疫荧光、逆转录聚合酶链反应(reverse transcription polymerase chain reaction， RT-PCR)和免疫蛋白印迹法(Western blotting)检测RPE细胞中DDR2的表达。缺氧条件下以Ⅰ型胶原(10μg/mL， 50℃孵箱内孵育)刺激，在刺激后即刻(即常氧状态下)、2，6，12，24h终止缺氧，用RT-PCR和Western blotting检测RPE细胞中DDR2和HIF-1α的表达，酶联免疫吸附试验(enzyme linked immunosorbent assay， ELISA)法观察RPE细胞培养上清中VEGF的表达。

结果：随着缺氧时间延长，RPE细胞中DDR2 mRNA和蛋白表达降低。在缺氧状态下Ⅰ型胶原仍可以随时间迁移使得DDR2 mRNA和蛋白表达增加，激活DDR2。缺氧胶原刺激组相对于缺氧组HIF-1α mRNA和蛋白表达减少，VEGF蛋白表达减少。

结论：缺氧条件下，RPE细胞中DDR2表达随时间推移逐渐降低。Ⅰ型胶原仍可以随时间迁移激活DDR2，缺氧胶原刺激可以抑制缺氧诱导的RPE细胞HIF-1α和VEGF表达上调。

AIM:To observe the expression of discoidin domain receptor 2(DDR2)in retinal pigment epithelium(RPE)cells under hypoxia state, the expression of DDR2, hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor(VEGF)in RPE cells under hypoxia state with stimulation of collagenⅠ, discussing the effects of collagenⅠ and DDR2 in choroidal neovascularization(CNV).

METHODS:It was an controlled experimental study, using 200μmol/L CoCl₂ treated RPE cells to establish chemical hypoxia model. The expression of DDR2 in cells was examined after hypoxia 0, 2, 6, 12, 24 hours by immunofluorescence, reverse transcription polymerase chain reaction(RT-PCR)and Western blotting. In hypoxia state with collagenⅠ's(10μg/mL in 50 degrees incubated box )stimulation, the expression of DDR2 and HIF-1α in cells were examined after hypoxia 0, 2, 6, 12, 24 hours by RT-PCR and Western blotting, using enzyme linked immunosorbent assay(ELISA)to observe the expression of VEGF in cell culture supernatant.

RESULTS:After prolonged hypoxia, the expression of DDR2 mRNA and protein were reduced. CollagenⅠcould multiply the expression of DDR2 mRNA and protein in hypoxia as time goes on, it could active DDR2. The expression of HIF-1α mRNA, protein and VEGF protein dropped off in hypoxia state with collagenⅠ's stimulation compared with hypoxia.

CONCLUSION: The expression of DDR2 in RPE cells reduced with time under hypoxia state. CollagenⅠcan activate DDR2 as time goes on. In hypoxia state with collagenⅠ's stimulation can surpress hypoxia induced up- regulation expression of HIF-1α and VEGF in RPE cells.

国家重点基础研究发展计划(973计划)资助项目(No.2011CB510200); 国家自然科学基金资助项目(No.81000392,81070748)