缺氧及血管内皮生长因子对视网膜色素上皮细胞Opticin蛋白表达的影响

The effect of vascular endothelial growth factor and hypoxia on the secretion of opiticin in retinal pigment epithelium cells

目的探讨缺氧及血管内皮生长因子（VEGF）对视网膜色素上皮（RPE）细胞Opticin蛋白表达的影响及其内在机制。方法实验研究。原代培养人眼RPE细胞，取生长良好的第3～6代细胞，接种于含DMEM培养液的6孔板中，于缺氧条件下或加入不同浓度（1、10、50及100μg／L）VEGF进行培养。逆转录聚合酶链反应（PCR）检测缺氧后RPE细胞中VEGFmRNA的表达。蛋白印迹法检测细胞内和细胞培养液中Opticin蛋白含量。明胶酶谱法检测细胞培养液中明胶酶活性。组间检测数据比较采用单因素方差分析。结果蛋白印迹法检测，显示缺氧组RPE细胞内Opticin蛋白含量无明显变化；而RPE细胞培养液中，缺氧组Opticin蛋白含量明显下降。逆转录PCR检测，缺氧组12、24hVEGFmRNA表达灰度值分别为0．81±0．04、0．67±0．07，均较正常对照组VEGFmRNA表达灰度值（0．21±0．03）明显升高，差异有统计学意义（F=483．60，P〈0．05）。添加不同浓度的外源性VEGF后，RPE细胞内Opticin蛋白表达无明显变化；添加1、10、50及100μg／L的外源性VEGF后，RPE细胞培养液中，Opticin蛋白表达灰度值分别为0．65±0．02、0．52±0．04、0．23±0．03、0．30±0．03，均较正常对照组Opticin蛋白表达灰度值（0．73±0．04）明显下降（F=141．38，P〈0．05）。明胶酶谱法分析，显示与基质金属蛋白酶-2相对应的位置出现明显的消化条带，且酶的活性随VEGF浓度的增加而递增。低浓度的乙二胺四乙酸可抑制由VEGF引起的RPE细胞培养液中Opticin蛋白含量减少。结论缺氧及VEGF作用可影响RPE细胞培养液中Opticin蛋白分泌，可能与增多的基质金属蛋白酶-2对Opticin蛋白的酶解作用有关。

Objective To evaluate the role of vascular endothelial growth factor or hypoxia on the secretion of opticin in retinal pigment epithelium cells. Methods Human RPE cells were cultured, the third to sixth passage of retinal pigment epithelium （RPE） ceils were placed in 6-well culture plates at a density of 4×10^4/well. For hypoxia experiment, the ceils were cultured under hypoxic condition for different times. For vascular endothelial growth factor （VEGF） experiment, the media was changed to DMEM containing different concentration VEGF （ 1, 10, 50, 100 μg/L） for 24 h respectively. VEGF mRNA levels were determined by RT-RCR method. The protein content of opticin in RPE cells or culture media was detected by Western blot. Matrix metalloproteinase activity in culture media was analysis by zomygraphy. One way ANOVA was used to test the comparisons between experimental groups and control group. Results Western blot experiment showed the opticin expression was not changed in RPE cells after hypoxia treatment, however was significantly decreased in culture media. Compared with control group （0. 21±0.03 ）. The relative density of VEGF mRNA levels （0. 81±0. 04, 0. 67±0. 07） in RPE cells were increased after 12 h or 24 h hypoxia treatment （ F = 483.60, P 〈 0. 05 ）. Opticin expression in RPE cells was also remain unchanged after vary concentration VEGF addition treatment （ F = 2. 16, P 〉 0. 05）, the relative density of opticin expression in VEGF conditioned culture medium were 0. 65 ±0.02,0. 52 ±0. 04,0. 23±. 03,0. 30±0. 03 respectively, and the difference in culture media was significant compared to control group （0. 73±0. 04） （F = 141.38, P 〈 0. 05 ）. Zomygrphagy indicate a matrix metalloproteinases type 2 digest band, the activities were enhanced with VEGF increasing. The decrease of opticin in culture media after VEGF treatment could be inhibited by low condition of EDTA. Conclusion VEGF and hypoxia have an effect on the on the secretion of opiticin in RPE cells, it may be contributed to the increasing levels of matrix metalloproteinases type 2.