色素上皮衍生因子在氧诱导血管增生性视网膜病变小鼠中的表达

The expression of pigment epithelium-derived factor in mice with oxygen-induced retinopathy

目的探讨氧诱导血管增生性视网膜病变小鼠视网膜中色素上皮衍生因子（PEDF）mRNA及蛋白的表达变化及意义。方法80只出生后2d的C57／BL6小鼠随机分为模型组（64只）和对照组（16只），出生后第7天模型组小鼠和母鼠一起放入氧气含量为75％的饲养箱中饲养，出生后第12天回到正常大气环境中饲养；对照组小鼠始终在正常大气环境中饲养。出生后第7、12、17天时，模型组及对照组在每个时间点分别取小鼠4只，荧光素心脏灌注后行视网膜铺片，荧光显微镜下观察血管分布和形态；提取模型组小鼠出生后第7、8、10、12、12．5、13、14、15、17、19天时间点的视网膜总RNA，通过逆转录-聚合酶链式反应（RT—PCR）检测PEDF、血管内皮生长因子（VEGF）、低氧诱导因子（HIF）-1α mRNA的表达变化；分别取模型组和对照组出生后第17天时的小鼠眼球，冰冻切片后通过免疫组织化学染色检测PEDF、VEGF、HIF-1α蛋白在视网膜组织中的表达变化。结果模型组小鼠在出生后第17天时视网膜有大量新生血管形成，视盘周围见大范围无灌注区PEDFmRNA表达在缺氧24h后开始下调，72h达到下降高峰，随后逐渐恢复正常水平,VEGF、HIF-1αmRNA则在高氧环境下表达下调，缺氧状态下迅速升高，于缺氧48h时到达高峰，然后缓慢下降；PEDF／VEGF、PEDF／HIF-1α比值在缺氧状态下显著降低。出生后第17天时，模型组小鼠视网膜中PEDF蛋白表达显著弱于对照组小鼠；而VEGF、HIF-1α蛋白表达则是模型组显著强于对照组。结论PEDFmRNA及蛋白在氧诱导血管增生性视网膜病变小鼠视网膜中明显下降，其趋势与VEGF和HIF-1α的变化相反，PEDF／VEGF、PEDF／HIF-1α表达平衡失调，可能是缺氧状态下视网膜新生血管生成的重要原因。

Objective To explore the changes and signifcance of expression of pigment epitheliumderived factor （PEDF） mRNA and protein in mice with oxygen-induced proliferative retinopathy. Methods Eighty 2-day-old C57/BL6 mice were randomly divided into model group （64 mice） and control group （16 mice）. On the 7th day after the birth, the mice in model group were raised in a box full of 75% O2 and then under the normal atmosphere condition on the 12th postnatal day. The mice in control group were raised under the normal circumstance all the time. On the 7th, 12th, and 17th day after birth, 4 mice in each group at each time point underwent fluorescein perfusion, and the vascular distribution and configuration were observed under the fluorescent microscope. The expression of PEDF.hypoxia-inducible factor （HIF）-1α and vascular endothelial growth factor （VEGF） mRNA in model group were detected by reverse transcription-polymerase chain reaction on the 7th, 8th, 10th, 12th, 12. 5th, 13th, 14th, 15th, 17th, and 19th postnatal day. The expressions of PEDF.HIF-1α and VEGF proteins in the 2 groups were dected by immunohistochemical staining on the 17th postnatal day. Results On the 7th day after the birth in the model group, retinal neovascularization and non-perfused area around the optic post were observed; the expression of PEDF mRNA decreased 24 hours after hypoxia, reached the peak at the 72nd hour, and increased to the normal level gradually; the expression of HIF-1α and VEGF mRNA decreased under the hyperoxia condition, increased under the hypoxia condition, reached to the peak at the 48th hour, and then decreased slowly; the ratio of PEDF/VEGF and PEDF /HIF-1α decreased markedly under the hypoxia condition. On the 17th postnatal day, the expression of PEDF protein in model group was weaker than which in control group; the expressions of HIF-1α and VEGF proteins in model group were more intensive than which in the control group. Conclusion The expressions of PEDF mRNA and protein in mice with oxygen-induced proliferative retinopathy decrease obviously, while VEGF and HIF-1α markedly increase. The balance maladjustment of the expression of PEDF/VEGF and PEDF/HIF-1α may be the important reason of hypoxic-induced retinal neovascularization.