缺氧和高浓度葡萄糖对体外培养人视网膜色素上皮衍生因子表达的影响

目的　探讨缺氧和高糖对体外培养的人视网膜色素上皮 (RPE)细胞血管内皮生长因子(VEGF)和色素上皮衍生因子 (PEDF)的影响。方法　我们通过实验性研究在氧正常或缺氧 (1%O2 )加或不加高浓度葡萄糖的条件下 ,培养的人RPE细胞 ,通过反转录PCR和实时定量分析检测PEDF和VEGF的表达 ,使用Western印迹分析检测PEDF和VEGF蛋白水平。结果　缺氧情况下 ,VEGF表达和蛋白水平增加 (P =0 .0 0 1) ;缺氧加 2 5mmol/L葡萄糖后增加更加明显 (P <0 .0 0 1)。缺氧情况下PEDF表达水平与正常氧环境比较无统计学意义 (P =0 .2 5 1) ;但加葡萄糖后PEDFmRNA表达水平低于正常对照。结论　体外培养的人RPE细胞在缺氧条件下PEDF表达降低不明显 ,这提示缺氧间接影响体外RPE细胞的PEDF下调 ,高葡萄糖直接下调PEDF的表达 ,同时增加VEGF的表达 ,这可能支持体内高血糖是糖尿病最危险的“糖毒性”观点。

Downregulation of the pigment epithelium derived factor by hypoxia and elevated glucose concentration in cultured human retinal pigment epithelial cells

Objective To explore the effect of hypoxia and high glucose concentration on the production and secretion of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF), an antiangiogenesis factor, in the cultured human retinal pigment epithelium (RPE) cells. Methods Human RPE cells were cultured under normoxic or hypoxic (1% O 2) condition with or without (25 mmol/L) glucose. RT-PCR and real-time quantificaiton analysis were used to examine the expression of VEGF and of PEDF mRNAs. Western blot analysis was used to measure the levels of VEGF and PEDF proteins. Results The expression of VEGF mRNA in RPE cells under hypoxic condition for 12 hours was 2.6 times that under the normoxic condition (P=0.001), and the expression of PEDF mRNA was only 0.77 time that of the controls (P=0.251). Under hypoxic condition with high concentration of glucose the expression of VEGF mRNA in RPE cells was 3.8 times that under the normoxic condition (P<0.001), and the expression of PEDF mRNA was further decreased (only 0.23 time that of the controls, P=0.02). The expression levels of VEGF protein under hypoxic condition with and without glucose were 1.69 and 1.27 times respectively those under the normoxic condition (P<0.0001 and P=0.004). The expression level of PEDF protein under hypoxic condition with glucose was 0.49 time that under the normoxic condition (P<0.000 1) and the expression level of PEDF protein under hypoxic condition without glucose was 0.92 time that under the normoxic condition (P=0.114). Conclusion Hypoxia indirectly influence the downregulation of PEDF and high concentration of glucose directly downregulates the expression of PEDF and increases the expression of VEGF simultaneously, thus supporting the concept that hyperglycemia is one of the most dangerous consequences of diabetes-associated "glucose toxicity" in vivo.