高糖对人脐静脉内皮细胞促红细胞生成素受体表达的影响

The effect of high glucose on the expression of erythropoietin receptor in human umbilical vein endothelial cells

目的探讨高糖条件下促红细胞生成素(EPO)受体mRNA及蛋白在人脐静脉内皮细胞(UVECs)的表达差异。方法体外常规培养人UVECs细胞株,实验组分别给予22 mmol/L葡萄糖作用12、24、48、72 h,5.5 mmol/L葡萄糖组(生理糖浓度)设为正常对照组,5.5 mmol/L葡萄糖+16.5 mmol/L甘露醇组为渗透压对照组。采用逆转录聚合酶链反应(RT-PCR)、免疫细胞化学法对高糖条件下人UVECs EPO受体mRNA和蛋白的表达进行检测。结果正常对照组和渗透压对照组相比,人UVECs EPO受体mRNA和蛋白表达差异均无统计学意义(P>0.05)。人UVECs在22 mmol/L高糖作用12、24、48、72 h后,EPO受体mRNA和蛋白表达均显著高于正常对照组(P<0.05),且随着时间的延长,EPO受体mRNA和蛋白的表达均逐渐增加。结论高糖条件下人UVECs EPO受体mRNA和蛋白表达均增强,且呈时间依赖性。人UVECs在22 mmol/L高糖条件下EPO受体mRNA及蛋白表达的增加与渗透压无关。

Objective Although vascular endothelial growth factor（VEGF） is a primary mediaor in diabetic retinopathy （DR）. VEGF inhibition alone is insufficient for preventing retinal neovascularization. Some studies showed that erythropoietin （EPO） is a potent retinal angiogenic factor of independent of VEGF in DR. The present study is to investigate the effect of high glucose on the expression of mRNA and protein of the erythropoietin receptor （EPOR） in cultured human umbilical vein endothelial cells（ UVECs） in vitro. Methods Human UVECs from the cell center of the hospital were cultured in vitro and passaged in DMEM containing 10% neonatal bovine serum with 22 mmol/L of glucose for 12,24,48 and 72 hours in the experimental group. Cells cultured in 5.5 mmol/L glucose were used as control group Ⅰ and mannitol ＋ 22 mmol/L of glucose （isotope） as control group Ⅱ. The expression of EPOR mRNA in Human UVECs were detected by semi-quantitative reverse transcriptase（RT）-polymerase chain reaction（PCR） and detected at A260mm/A280mm. The PCR product was calculated as the A value of EPOR mRNA amplification/the A value of GAPDH mRNA amplification. The expression of EPOR protein in Human UVECs was detected by immunoeytochemistry. Results The A260mm/A280mm, value of EPOR mRNA receptor expression was 0.32 ±0.02 in the 5.5 mmol/L glucose group, and 0.34 ± 0.02 in the mannitol ＋ 22 mmol/L glucose group（P 〉 0.05）. In 12 hours,24 hours and 72 hours after the experiment, the A260mm/A280mm value of EPOR mRNA was 0. 82 ± 0.01,0. 96± 0.02 and 1.02 ±0.01 ,respectively,indicating a significant increase in comparison with the 5.5 mmol/L glucose group. The expression of Human UVECs protein was gradually increased with passage in the experimental group. Expression of Human UVECs protein was stronger in various time points in the 22 mmol/L glucose group than in the 5.5 mmol/L glucose group. Conclusion High glucose elevates the expression of EPOR mRNA and protein in Human UVECs in a time-dependent manner. The effect of high glucose（22 mmol/L glucose）on the expression of EPOR mRNA and protein in Human UVECs is not related to osmotic pressure.