视网膜酸化诱导VEGF与PEDF表达及其与氧化应激的关系

Regulation of vascular endothelial growth factor and pigment epithelium-derived factor in rat retinal explant under the retinal acidification

背景缺氧与高糖是引起视网膜新生血管生长的主要原因，可引起视网膜糖酵解作用增强，导致组织酸中毒。目的探讨视网膜酸中毒对视网膜血管内皮生长因子（VEGF）与色素上皮衍生因子（PEDF）表达的影响及氧化应激的作用。方法从2周龄雄性SD大鼠中分离出视网膜。分别在NaHCO，调制的pH7．2、6．8、6．5酸性环境的DMEM培养液中培养24h；另外，在上述酸性培养液中培养后用PBS洗涤视网膜2遍后置于pH值为7．2的新鲜培养液中继续培养24h；同时，在上述酸性培养液中加入抗氧化剂对视网膜进行培养。制作视网膜标本，然后行苏木精-伊红染色，采用荧光定量聚合酶链反应（PCR）及免疫蛋白印迹技术检测各组大鼠视网膜中VEGF和PEDF蛋白及其mRNA的表达，以pH7．2作为对照。结果视网膜培养24h后，pH7．2组、pH6．8组视网膜层次清晰，但pH6．5组视网膜出现空泡。正常视网膜VEGFmRNA的表达为（112±11）％，pH7．2组为（100±7）％，差异无统计学意义（P=0．55）；pH6．8组、pH6．5组中的视网膜VEGFmRNA分别为（196±43）％、（251±29）％，均较pH7．2组明显升高，差异均有统计学意义（P〈0．05）。正常视网膜PEDFmRNA水平为（86±19）％，pH7．2组为（100±33）％，差异无统计学意义（P=0．64）；pH6．5组视网膜PEDFmRNA水平为（230±66）％，较pH7．2组明显升高（P〈0．05）。VEGF与PEDF蛋白与其mRNA表达趋势一致。视网膜酸化纠正后，pH7．2、6．8、6．5组视网膜VEGFmRNA分别为（100±13）％、（111±9）％、（113±9）％，3个组之间比较差异无统计学意义（F=2．51，P=0．16）。PEDFmRNA的表达分别为（100±13）％、（110±9）％、（108±11）％，3个组之间比较差异无统计学意义（F=0．98，P=0．43）。加入抗氧化剂后，pH7．2组视网膜VEGFmRNA水平为（100±9）％，pH6．8组为（106±7）％，pH6．5组为（148±22）％，pH6．5组VEGFmRNA表达水平均明显高于pH7．2组和pH6．8组（P〈0．05）。pH7．2、6．8、6．5组大鼠视网膜PEDFmRNA表达分别为（100±31）％、（282±45）％、（480±117）％，差异有统计学意义（F=20．73，P=0．00）。结论视网膜酸化诱导VEGF的表达受到氧化应激的调节，抗氧化剂可促进酸化视网膜PEDF的表达增加，表明氧化应激可抑制PEDF表达。

Background Hypoxia and hyperglycemia are the common causes of retinal neovascularization. In these states, H＋ accumulates because of the elevated glycolysis and failure of retinal circulation, thus the retinas readily acidified. Objective The present study was to explore whether retinal acidosis independently regulates the production of vascular endothelial growth factor （VEGF） and pigment epithelium-derived factor （ PEDF ） and whether the regulation is related to oxidative stress. Methods The retinas from 2-week-old male SD rats were cultured with explant method in DMEM modulated by NaHCO3 ,and culture retinas were randomly divided into pH 7.2,6.8 and 6.5 groups for 24 hours. In addition,after 24 hours of culture as above described,retinas were washed using PBS two times and then followed by again culture in DMEM with pH 7.2 for another 24 hours. Also,antioxidant was added in different pH values of DMEM for culture as above described. The retinal samples were prepared for histopathological examination. The expressions of VEGF and PEDF proteins and their mRNA in retina tissue were detected by Western blot and fluorescence quantitative polymerase chain reaction （PCR） respectively. Results The retina showed the clear structure and morphology in pH 7.2 group and pH 6.8 group, but retinal vacuoles change was seen in pH 6.5 group after culture for 24 hours. No significant difference was seen in the expressing level of VEGF mRNA in retina between normal group and pH 7.2 group（112%±11% vs 100%＋7%）（ P=0.55）,but those in pH 6.8 group and pH 6.5 group were significant increased in comparison with pH 7.2 group（ 196% ±43% vs 100%±7% ;251%±29% vs 100%±7% ） （ P〈0.05 ）. The expressing level of PEDF mRNA in retina in normal group was similar to that of pH 7.2 group（ 86% ±19% vs 100%±33% ）（P = 0.64）, but that in pH 6.5 group was significantly higher than pH 7.2 group （230%±66% vs 100%±33 % ） （P〈0.05）. The resemble results were found in the expressions of V EGF and PEDF protein. After pH reversion, the expressing levels of VEGF mR NA were 100%±13 % , 111%±9% , 113%±9% in pH 7.2 group, pH 6.8 group and pH 6.5 group respectively without significant difference among them （F=2.51,P=0.16）. The expressing levels of PEDF mRNA were 100%±13%, 110% ±9%, 108% ±11% in different groups （ F= 0.98, P = 0.43 ）. Under the presence of antioxidant, the expressing level of VEGF mRNA in pH 6.5 group increased in comparison with pH 7.2 group and pH 6.8 group（P〈0.05 ）. The expressing levels of PEDF mRNA were significant different among pH 7.2 group （100±31）% , pH 6.8 group （282±45）% and pH 6.5 group（480±l17）% （F=20.73,P=0.00）. Conclusions VEGF can be induced by retinal acidification alone, which may be regulated by oxidative stress. Under the retinal acidification, antioxidants promote the expression of PEDF,suggesting that oxidative stress inhibits the production of PEDF.