【摘要】 目的制备血氧浓度波动所致新生大鼠增生性视网膜病变的模型,进一步探讨早产儿视网膜病(ROP)的发病机制。方法 208只新生 SD 大鼠,随机分为氧气组和对照组,采用免疫组织化学方法以及 RT-PCR 技术,观察生后14、18、25 d 视网膜组织中血管内皮生长因子(VEGF)及 Flk-1(VEGFR-2)蛋白和 mRNA 表达水平。并且在氧气暴露后4 d,取幼鼠眼球进行视网膜铺片以及组织石蜡切片 HE 染色,观察视网膜血管的改变情况。结果 (1)氧气组18 d 幼鼠视网膜无血管面积明显大于空气组(P<0.05),且突破内界膜的内皮细胞核数目明显多于空气组(P<0.05),表明模型建立成功。(2)氧气组视网膜 VEGF 及 Flk-1在14、18 d 时表达均明显强于对照组(P<0.05),在25d时阳性表达明显减弱,与对照组比较,差异无统计学意义(P>0.05)。且 VEGF 及 Flk-1在18 d 时表达最强,与14及25 d 比较,表达强度差异均有统计学意义(P<0.05)。(3)氧气组视网膜组织 VEGFmRNA 及 Flk-1 mRNA 表达水平在14 d、18 d 均明显高于对照组(P<0.05)。25 d 时氧气组 VEGFmRNA 及 Flk-1 mRNA 表达水平均下降,与对照组比较,差异无统计学意义(P>0.05)。结论反复血氧浓度的波动可导致新生大鼠视网膜血管增生性病变,血管增生性视网膜组织中 VEGF 及 Flk-1表达明显增强。本研究还提出,ROP 视网膜血管增生过程中存在一种正反馈机制即 VEGF 与 Flk-1的上调协同作用,共同促进视网膜新生血管的形成。更多还原

【Abstract】 Objective Fluctuations in arterial oxygen are associated with development of severe retinopathy of prematurity（ROP）in humans.However,the causal relationship between oxygen variability and ROP remains unknown.The authors developed a rat model of retinal neovascularization by repeated fluctuations of inhaled oxygen between hypoxia and hyperoxia to investigate the mechanism of the development of retinal neovascularization,the regulation of vascular endothelial growth factor（VEGF）and KDR/Flk-1（VEGFR-2）expression.Methods Two hundred and eight newborn Sprague Dawley rats were randomly divided into oxygen and air groups.The oxygen concentration in the oxygen group was alternated between 50% and 10% every 24 hours for 14 days.The control group were kept in room air environment. VEGF and Flk-1 expression was observed at 14,18 and 25 days after birth in both exposed group and control group by immunohistochemical staining and semiquantitative RT-PCR.The status of retinal vasculature on day 4 after oxygen exposure was also observed.The retinas were dissected and stained by using a histochemical method for detecting adenosine diphosphatase（ADPase）activity,digital images of the retinas were captured and the peripheral avascular retina were measured.HE staining on methacrylate sections of eyes was used for counting the number of nuclei extending from retinal area into vitreous to identify extraretinal neovascularization.Numeric data were expressed as the mean±standard deviation（SD）. Statistical calculations were performed using the SAS 8.1 statistical package.Differences in measured variables between experimental and control groups were determined using comparison of the means using two- way analysis of variance（ANOVA）statistical calculations and T-test.AP value less than 0.05 was regarded as significant.Results（1）The animal model was successfully established:the avascular areas of retina of 18-day-old rats were larger than those of the control group and the numbers of nuclei extending from retinal area into vitreous in exposed group were significantly higher compared to the control（P<0.05）.（2）The expression of VEGF and Flk-1 on the 14^thday in the oxygen group was significantly stronger than that of the control group（P<0.05）.In the oxygen group,VEGF and Flk-1 expression was the strongest in the retina on the 18^thday,the result had significant difference as compared with the 14^thand 25^thday（P<0.05）,and they were also stronger than that of the control group（P<0.05）.The expression of VEGF and Flk-1 decreased on the 25^thday and had no significant difference as compared with the control group（P>0.05）. （3）Both VEGF-mRNA and Flk-1-mRNA significantly increased on the 14^thday and the 18th day（P<0. 05）.On the 25^thday,the amounts of VEGF-mRNA and FIk-1-mRNA were similar between the control and oxygen group（P>0.05）.Conclusion Fluctuation in oxygen is associated with the development of retinal neovascularization in the retinopathy.Increased expressions of VEGF and Flk-1 in the oxgen fluctuations- induced neovascularized retina suggested that VEGF and Flk-1 might play a critical role in the pathogenesis of ROP.The results also indicated the positive feedback in the pathogenesis of ROP that the synergistic interaction of VEGF and Flk-1 in the retinal vascular proliferation.These findings provide insight into the effect of repeated oxygen fluctuation on the development of severe ROP in preterm infants.更多还原