GDF-15在高糖缺氧环境下人视网膜色素上皮细胞中的表达及意义

Expression of GDF-15 in Human Retinal Pigment Epithelial Cells in High Glucose and Hypoxia Environment

目的观察生长分化因子15(Growth differentiation factor-15,GDF-15)在模拟高糖缺氧环境下人视网膜色素上皮细胞中的表达,探讨姜黄素对其表达的影响。方法将培养的人视网膜色素上皮(human retinal pigment epithelial cells,hRPE)细胞分为正常对照组(5 mmol/L葡萄糖)、高糖缺氧组(25 mmol/L葡萄糖+200μmol/L CoCl_(2))和高糖缺氧加药组(25 mmol/L葡萄糖+200μmol/L CoCl_(2)+20 ng/ml姜黄素)。采用MTT法检测各组细胞增殖率;采用免疫荧光染色法检测各组细胞中GDF-15和血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)的表达及其定位;采用RT-PCR法检测各组细胞中GDF-15和VEGF mRNA的表达;采用Western blot法定量检测各组细胞中GDF-15和VEGF蛋白的表达。结果MTT检测显示高糖缺氧组细胞增殖率大于正常对照组,高糖缺氧加药组细胞增殖率低于缺氧组,差异均具有统计学意义(P<0.001)。免疫荧光检测结果显示,正常对照组细胞中GDF-15和VEGF均呈弱性表达,高糖缺氧组细胞中GDF-15和VEGF均呈强阳性表达,高糖缺氧加药组细胞中GDF-15和VEGF表达较高糖缺氧组减弱,GDF-15及VEGF表达位点基本一致。实时荧光定量PCR法检测显示高糖缺氧组GDF-15和VEGF的mRNA相对表达量较正常组与高糖缺氧加药组mRNA相对表达量升高,差异均具有统计学意义(P<0.001)。Western blot法检测显示高糖缺氧组GDF-15和VEGF的蛋白相对表达量均明显高于正常对照组和高糖缺氧加药组,差异均具有统计学意义(P<0.001)。结论体外高糖缺氧环境下可以诱导人视网膜色素上皮细胞中GDF-15表达上调,姜黄素可抑制高糖缺氧环境下视网膜色素上皮细胞的增殖及GDF-15的过表达。

Objective To observe the expression of GDF-15 in high glucose and hypoxia hRPE cells and the effect of curcumin on it.Method hRPE cells were divided into normal control group,high glucose+hypoxia group and high glucose+hypoxia plus drug group.Immunofluorescence staining was used to detect the expression and localization of GDF-15 and VEGF;RT-PCR was used to detect GDF-15 and VEGF mRNA;Western blot was used to detect GDF-15 and VEGF protein.Results MTT assay showed that the cell proliferation rate in the high glucose hypoxia group was greater than that in the normal control group,and the cell proliferation rate in the high glucose hypoxia plus drug group was lower than that in the hypoxia group,all differences were statistically significant(P<0.001).Immunofluorescence assay showed that both GDF-15 and VEGF were weakly expressed in cells from the normal control group,that both GDF-15 and VEGF were strongly positively expressed in cells from the high glucose hypoxia group,that the expression of GDF-15 and VEGF in cells from the high glucose hypoxia plus drug group was attenuated compared with that in the hypoxia group,and that the sites of GDF-15 and VEGF expression were basically the same.Real time PCR assay showed that the mRNA relative expression of GDF-15 and VEGF in the high glucose hypoxia group was increased compared with that in the normal group and hypoxia plus drug group,and the differences were all statistically significant(P<0.001).Western blot assay showed that the protein relative expressions of GDF-15 and VEGF in the high glucose hypoxia group were significantly higher than those in the normal control group and the high glucose hypoxia plus drug group,and the differences were all statistically significant(P<0.001).Conclusions High glucose and hypoxia can upregulate the expression of GDF-15 in hRPE cells,and curcumin inhibits the overexpression of GDF-15.