CREB1在氧诱导视网膜病变小鼠模型中的表达变化

目的研究CREB1在氧诱导视网膜病变（OIR）小鼠模型视网膜新生血管形成中的表达变化,探寻视网膜新生血管形成的可能机制。方法实验研究。选用7日龄健康清洁级C57BL/6J小鼠134只,随机分为正常对照组（67只）和OIR模型组（67只）。将小鼠与哺乳母鼠共同置于（75±2）％氧环境内饲养5 d后转移至正常环境中饲养5 d,建立小鼠OIR模型,17日龄的OIR鼠在空气中再继续饲养4 d,正常对照组在正常氧环境中饲养21 d。出生后第17 d时取OIR模型组和正常对照组小鼠制备视网膜切片HE染色法和FITC-dextran心脏灌注视网膜铺片法观察视网膜新生血管情况,以及视网膜组织冰冻切片免疫荧光染色观察P-CREB1表达情况。取2组鼠第7、9、12、14、17、21 d的视网膜采用实时定量聚合酶链反应（real-time PCR）法和Western Blot法检测CREB1mRNA和蛋白在小鼠视网膜中的表达情况。数据分析采用独立样本t检验和两因素方差分析方法,两两比较采用Bonferronipost-test检验。结果17 d时OIR模型组较正常对照组突破视网膜内界膜的内皮细胞核数明显增加（t=11.31,P<0.05）,且模型组视网膜新生血管区及无灌注区面积分别为（21.40±2.72）%和（30.61±3.12）%。与正常对照组相比,模型组小鼠视网膜中可见大量P-CREB1蛋白荧光,主要表达在内核层和神经节细胞层。Real-time PCR和Western Blot结果表明,正常对照组第7、9、12、14、17天的CREB1mRNA和蛋白在视网膜中的表达水平逐渐升高,21 d时开始出现下降的趋势;在各相应时间点OIR模型组里的CREB1相对表达量除第7天外,其余时间点均高于正常对照组,差异均有统计学意义（P<0.05）。结论CREB1的表达水平与视网膜新生血管的形成存在时空对应关系,CREB1的高表达可能参与了OIR模型中视网膜新生血管的形成过程。     

DNA损伤标志物γH2AX在氧诱导视网膜新生血管形成中的作用

背景 氧浓度变化诱导产生视网膜新生血管,并导致细胞DNA损伤反应.以往认为促血管生成因子过度表达是血管新生的原因,但DNA损伤是否与视网膜新生血管有关尚不清楚. 目的 探讨参与DNA损伤修复的磷酸化蛋白γH2AX与小鼠视网膜血管新生的关系. 方法 应用72只17日龄C57BL/6J小鼠按照随机数字表法随机分为正常对照组、氧诱导视网膜病变(OIR)模型组、OIR阳性对照组和OIR阴性对照组,每组18只.视网膜铺片免疫荧光法对比观察视网膜新生血管形态学变化及γH2AX表达情况.将人脐静脉血管内皮细胞(HUVECs)分为正常对照组、低氧模型对照组、阳性干扰组和阴性干扰组.细胞处理后12h,采用免疫荧光法比较并分析各组γH2AX的表达情况,采用Western blot法定量检测各组γH2AX的表达. 结果 正常对照组、OIR模型组、OIR阳性对照组和OIR阴性对照组17日龄小鼠视网膜新生血管面积和无灌注区面积比较,总体差异均有统计学意义(F=437.62、93.05,均P＜0.01),其中OIR模型组和OIR阴性对照组小鼠视网膜新生血管面积和无灌注区面积均较正常对照组明显增大;OIR阳性对照组小鼠视网膜新生血管面积和无灌注区面积均较OIR模型组和OIR阴性对照组明显减小,差异均有统计学意义(均P＜0.01).17日龄小鼠视网膜γH2AX焦点团聚集出现情况与新生血管和无灌注区面积大小变化趋势相一致.正常对照组、低氧模型对照组、阳性干扰组和阴性干扰组HUVECs中γH2AX焦点阳性细胞百分数比较,差异有统计学意义(F=64.97,P＜0.01),其中低氧模型对照组和阴性干扰组γH2AX焦点阳性细胞百分数均较正常对照组明显增大,差异均有统计学意义(均P＜0.01);阳性干扰组均较低氧模型对照组和阴性干扰组明显减小,差异均有统计学意义(均P＜0.01).各组HUVECs间γH2AX的表达量与免疫荧光趋势相一致.结论 缺氧环境下,γH2AX在小鼠视网膜血管和体外培养的HUVECs中均大量表达.γH2AX的表达与视网膜血管新生有关.低氧诱导的DNA损伤修复反应可能与视网膜血管新生有一定关系.     

ILK在氧诱导视网膜病变小鼠模型视网膜组织中的表达

背景 研究表明,整合素连接激酶( ILK)在新生血管形成过程中发挥重要作用,目前ILK在其他组织器官新生血管形成过程中的机制已有报道,但少有其与视网膜新生血管形成关系的研究. 目的 探讨ILK在氧诱导视网膜病变(OIR)小鼠模型视网膜新生血管形成和退化过程中的表达及其意义.方法 选用7日龄健康清洁级C57BL/6J小鼠128只,按随机数字表法分为正常对照组和OIR模型组.将小鼠与哺乳母鼠共同置于体积分数(75±2)％氧的玻璃氧箱内饲养5d后转移至正常环境中饲养5d,建立小鼠OIR模型,正常对照组在正常氧环境中饲养21d.取OIR模型组和正常对照组出生后第17天小鼠各5只制备视网膜切片,进行苏木精-伊红染色,检测每张切片中突破视网膜内界膜的血管内皮细胞核数目.取出生后第12、14、17、21天OIR模型组和正常对照组小鼠各4只分别制备视网膜切片和视网膜铺片,应用ADP酶组织化学法动态观察视网膜新生血管形成和退化过程;采用免疫组织化学法、实时定量聚合酶链反应( real-time PCR)法和Western blot法检测ILK蛋白及其mRNA在小鼠视网膜中的表达情况. 结果 OIR模型组突破视网膜内界膜的血管内皮细胞核计数为(45.64±12.17)个,正常对照组为(0.35±0.14)个,两组间差异有统计学意义(t=22.85,P＜0.05).视网膜铺片结果显示,OIR模型组小鼠第14天视网膜新生血管开始出现,与同龄正常对照组小鼠视网膜血管比较,出生后17d小鼠新生血管形成和血管走行异常等达到高峰,至21d时新生血管开始退化.免疫组织化学检测显示,ILK主要表达于视网膜神经节细胞层、内核层、内丛状层和光感受器层.Real-time PCR和Western blot结果表明,OIR模型组第12、14、17、21天ILK mRNA在视网膜中的表达水平为(1.00±0.22)、(1.85±0.17)、(1.58±0.43)、(1.53±0.36),呈先升高后下降的趋势;在第12、14、17、21天与正常对照组比较小鼠OIR模型组ILK/3-actin吸光度值均高于正常对照组,差异均有统计学意义(t=2.97,P＜0.05;t=11.88,P＜0.01;t=16.84,P＜0.01;t=13.00,P＜0.01). 结论 ILK的表达水平与视网膜新生血管的形成存在时空对应关系,ILK的高表达可能与视网膜新生血管形成密切相关.     

玻璃体腔注射VAS2870对小鼠氧诱导视网膜病变的保护作用

目的：观察玻璃体腔注射VAS2870对C57小鼠氧诱导视网膜病变的影响。方法：将新生C57 BL/6 J小鼠随机分为3组,分别为正常对照组、VAS2870注射 OIR 组和无菌 PBS 缓冲液注射OIR组。将后两组小鼠在出生后第7 d ( P7)至P12置于体积分数为75%±2%的恒定高氧氧箱中以构建OIR模型,在 P12时给予幼鼠双眼玻璃体腔注射 VAS2870(0.5μL),另一组幼鼠双眼注射同等剂量的无菌PBS缓冲液。三组小鼠均在 P17时取右眼行视网膜铺片和Lectin染色,观察视网膜中央无血管区及病理性新生血管的情况;取右眼行视网膜组织定量检测 ROS/RNS 含量;取左眼应用RT-PCR检测Nox4 mRNA含量,并应用Western-blot测定视网膜组织中VEGF的表达。结果：VAS2870注射OIR组小鼠视网膜中央无血管区面积较无菌PBS缓冲液注射OIR组明显减少( P<0.05),病理性新生血管数目明显减少( P<0.05);VAS2870注射OIR组小鼠视网膜组织Nox4 mRNA的表达量明显低于无菌PBS缓冲液注射组;VAS2870注射OIR组小鼠视网膜组织ROS含量较无菌PBS缓冲液注射组明显降低( P<0.05);VAS2870注射OIR组小鼠视网膜组织VEGF的表达明显低于无菌PBS缓冲液注射组( P<0.05)。结论：在小鼠OIR模型中, VAS2870可抑制Nox4 mRNA的表达,减少ROS/RNS,下调VEGF的生成,在视网膜病变进程中具有保护作用。     

基质细胞衍生因子-1在氧诱导视网膜病变小鼠视网膜中的表达

背景早产儿视网膜病变（ROP）的进展与多种新生血管调控因子有关,而基质细胞衍生因子·1（SDF．1）在氧诱导视网膜病变（OIR）动物模型视网膜中的表达及其作用机制尚不明确。目的对SDF-1在OIR小鼠模型视网膜中的表达进行定位和定量分析。方法应用随机数字表法将动物随机分组。将20只7日龄C57BL／6J幼鼠暴露在体积分数（75±2）％浓度的高氧状态下5d,随后在正常氧环境下5d,作为OIR组;另20只同日龄幼鼠作为正常对照组。通过免疫组织化学染色和实时荧光定量聚合酶链反应（real-timePCR）法观察视网膜的SDF-1的蛋白表达以及SDF-1mRNA的变化。结果OIR组12日龄小鼠的视网膜神经节细胞层可见SDF．1蛋白呈阳性表达;OIR组17习龄小鼠的神经节细胞层、内层视网膜的血管内皮细胞、新生血管内皮细胞可见SDF-1蛋白呈强阳性表达;正常对照组12日龄小鼠SDF-1蛋白和正常对照组17日龄小鼠SDF-1蛋白微弱表达于内层视网膜及视网膜血管附近。OIR组17日龄小鼠的SDF-1mRNA表达水平明显高于OIR组12日龄小鼠（P〈O．01）及正常对照组17日龄小鼠（P〈0．01）;OIR组12日龄小鼠SDF-1mRNA表达水平明显低于正常对照组12日龄小鼠（P〈0．05）。OIR组17日龄小鼠的SDF-1mRNA表达水平明显高于OIR组12日龄小鼠（t=8．072,P〈0．05）和正常对照组17日龄小鼠（t=10．026,P〈O．05）;OIR组12日龄小鼠SDF-1mRNA表达水平明显低于正常对照组12日龄小鼠（t=4．336,P〈0．05）。结论SDF-1在相对低氧状态下的视网膜中表达明显上调,因而可促进OIR的视网膜新生血管形成。     

Spatial pattern and temporal evolution of retinal oxygenation response in oxygen-induced retinopathy

PURPOSE: To determine the spatial pattern and temporal evolution of the change in retinal partial oxygen pressure (DeltaPO(2)) associated with a murine oxygen-induced retinopathy (OIR) model of retinal neovascularization (NV). METHODS: On P7, newborn C57BL/6 mice were exposed to 75% oxygen until postnatal day (P)12, followed by recovery in room air until P17 or P34. Control mice remained in room air until P17 or P34. At P17 and P34, functional magnetic resonance imaging (MRI) and a carbogen inhalation challenge was used to measure retinal DeltaPO(2). Retinal avascularity, distance from the optic nerve head to the vascular edge in the peripheral retina, and NV incidence and severity were measured in retinas stained with adenosine diphosphatase (ADPase). RESULTS: In P17 and P34 controls and in P34 OIR animals, retinas were fully vascularized without evidence of NV. In P17 OIR mice, there was a large central retinal capillary-free zone (22% +/- 3% of the entire retinal area, mean +/- SD) and 4 clockhours (range 1-7) of retinal NV at the border of the peripheral vascular and central acapillary retina in 100% (36/36) of the mice. In P17 OIR mice, retinal DeltaPO(2) over the vascularized far peripheral retina was not significantly (P > 0.05) different from the P17 control but was supernormal (P < 0.05) over the central capillary-free retina. However, no differences (P > 0.05) in retinal DeltaPO(2) were found between the P34 control and OIR groups. CONCLUSIONS: A reversible supernormal DeltaPO(2) was found only over the central acapillary retina during the appearance of retinal NV in a mouse OIR model. The present data show the applicability of carbogen-challenge functional MRI to the study of retinal DeltaPO(2) in vivo in eyes that are too small for the use of existing techniques.     

高迁移率族B1(HMGB-1)蛋白在鼠视网膜新生血管形成中的调控作用

目的　探讨高迁移率族Ｂ１（ｈｉｇｈｍｏｂｉｌｉｔｙｇｒｏｕｐｂｏｘ-１,ＨＭＧＢ-１）蛋白在鼠视网膜新生血管形成中的调控作用。方法　将基质胶（Ｍａｔｒｉｇｅｌ）和ＨＭＧＢ-１混合物注入鼠龄为７ｄ（Ｐ７）的Ｃ５７ＢＬ／６Ｊ健康小鼠左眼视网膜下,基质胶与ＰＢＳ混合物注入右眼球作为对照组。注入１０ｄ后对获得的视网膜组织切片进行ＨＥ染色,计数突破视网膜前膜的血管内皮细胞核数。选用Ｃ５７ＢＬ／６Ｊ健康新生鼠建立氧诱导视网膜病变（ｏｘｙｇｅｎ-ｉｎｄｕｃｅｄｒｅｔｉｎｏｐａｔｈｙ,ＯＩＲ）动物模型,出生后第７天（Ｐ７）放入高氧环境中连续饲养５ｄ,然后置入正常空气中继续饲养;正常空气对照组小鼠则在空气中饲养。在Ｐ１７采用ＤｉＩ荧光造影视网膜铺片观察ＯＩＲ模型组和正常空气对照组小鼠视网膜血管形态变化;同时采用Ｗｅｓｔｅｒｎ-ｂｌｏｔ检测模型组和对照组小鼠玻璃体中ＨＭＧＢ-１蛋白的表达水平。结果　ＨＭＧＢ-１基质胶模型组与ＰＢＳ基质胶对照组基质胶区域突破视网膜内界膜的血管内皮细胞核数分别为（４２．１９±１．７６）个、（３１．２４±１．２５）个,经过ＨＭＧＢ-１与基质胶混合物处理的眼球中新生血管水平显著高于对照组（ｔ＝－４２．４２,Ｐ＝０. ０００）。ＤｉＩ荧光造影视网膜铺片结果显示,ＯＩＲ模型组鼠视网膜自视盘向中周部出现大片无灌注区,无灌注区周围出现新生血管丛;Ｗｅｓｔｅｒｎ-ｂｌｏｔ分析显示,与正常空气对照组相比,ＯＩＲ模型组鼠玻璃体中ＨＭＧＢ-１蛋白表达增加了７８％ ±７％,出现了显著上调（ｔ＝－４７．９０,Ｐ＝０．０００）。结论　ＨＭＧＢ-１在视网膜新生血管形成中起重要的调控作用。     

夜间光照对氧诱导的视网膜病变小鼠视网膜新生血管形成的影响

背景 氧诱导的视网膜新生血管形成是多种视网膜血管性疾病的病理学基础,预防视网膜新生血管的形成可缓解视网膜病变对视网膜的损害程度.研究表明夜间睡眠时给予光照可能对早期糖尿病视网膜病变(DR)患者有利,但其对早产儿视网膜病变(ROP)患者视网膜新生血管有无影响报道较少.目的 观察夜间光照对氧诱导视网膜病变(OIR)小鼠视网膜新生血管形成的影响.方法 将64只SPF级新生C57BL/6J小鼠按随机数字表法随机分为正常对照组、单纯夜间光照组、OIR模型组、OIR联合夜间光照组,每组16只小鼠.正常对照组和单纯夜间光照组小鼠生长于正常空气(氧体积分数21％);OIR模型组和OIR模型联合夜间光照组小鼠于出生后第7天置于高氧环境(75％±2％)生长,出生第12天调整氧体积分数为正常;OIR联合夜间光照组和单纯夜间光照组于出生后第12～17天给予夜间光照,光照度为100 Ix.各组小鼠均于出生后第17天摘除眼球,采用ADP酶法制备视网膜铺片,了解视网膜血管的改变情况;视网膜组织切片行苏木精-伊红染色并计数突破视网膜内界膜的新生血管内皮细胞核数;免疫组织化学法观察各组小鼠视网膜中血管内皮生长因子(VEGF)的表达,实时荧光定量聚合酶链反应(PCR)法检测各组视网膜中VEGFmRNA的表达.实验动物的使用和喂养遵循ARVO声明.结果 正常对照组和单纯夜间光照组小鼠视网膜血管形态均无明显差异.OIR模型组视网膜铺片显示视网膜中央部大片无血管区,大量结构异常的新生血管形成.与OIR模型组相比,OIR模型联合夜间光照组视网膜中央无血管区面积以及新生血管分布密度减少.在实验后第17天时,正常对照组和单纯夜间光照组视网膜新生血管内皮细胞核数分别为(0.97±0.83)个和(1.00±0.72)个,OIR模型组为(38.57±5.01)个,而OIR联合夜间光照组小鼠视网膜新生血管内皮细胞核数为(16.92±3.39)个,总体差异有统计学意义(F=78.767,P=0.000),OIR联合夜间光照组视网膜新生血管内皮细胞核数明显少于OIR模型组,差异有统计学意义(t=20.446,P＜0.01).免疫组织化学法检测显示OIR模型联合夜间光照组中VEGF蛋白表达明显少于OIR模型组.正常对照组、单纯夜间光照组、OIR模型组和OIR联合夜间光照组小鼠视网膜VEGF mRNA相对表达量分别为1.00±0.00、0.94±0.07、2.08±0.50和1.43±0.21,各组间的总体差异有统计学意义(F=11.268,P=0.003),OIR模型联合夜间光照组表达较OIR模型组下调,差异有统计学意义(t=20.163,P＜0.05).结论 夜间光照可减少OIR小鼠视网膜新生血管的形成.     

HIF-2alpha-haploinsufficient mice have blunted retinal neovascularization due to impaired expression of a proangiogenic gene battery

PURPOSE: To characterize the effect of HIF-2alpha haploinsufficiency on retinal neovascularization and angiogenic signaling in neonatal mice. METHODS: Retinal samples were obtained from HIF-2alpha-haploinsufficient (Epas1+/-) and wild-type (Epas1+/+) neonatal mice subjected to an oxygen-induced retinopathy (OIR) protocol. Histologic and molecular studies were performed immediately, 12 hours, or 5 days after initiation of the hypoxia phase of the OIR protocol. Molecular profiling was performed in mouse brain endothelial cells maintained in normoxia or hypoxia. Transfection studies assessed the response of isolated promoter regions from proangiogenic genes to HIF-1alpha or -2alpha overexpression. RESULTS: Epas1+/- mice exhibited no significant differences in retinal vasculature during normal development but had reduced retinal neovascularization in an OIR protocol. Multiple proangiogenic factors were induced during the hypoxia phase in Epas1+/+ OIR retinal samples, whereas Epas1+/- OIR retinal samples had absent or blunted induction of these same factors. Several, but not all, proangiogenic factors were induced in mouse brain endothelial cells after hypoxia. In transfection assays, most proangiogenic promoter regions were preferentially activated by HIF-2alpha relative to HIF-1alpha. CONCLUSIONS: HIF-2alpha deficiency results in reduced neovascularization and blunted inducibility of multiple proangiogenic factors in the retinas of mice with OIR. The authors propose that HIF-2alpha is a master regulator of proangiogenic factors in retinal vascular endothelial cells, the predominant cell type of the retina in which HIF-2alpha is expressed. Future studies will address whether the molecular and functional roles for HIF-2alpha identified from these studies can be generalized to other pathophysiological states involving neovascularization.     

巨噬细胞在小鼠氧诱导视网膜新生血管形成中的作用

目的　探讨巨噬细胞对小鼠氧诱导视网膜病变（ｏｘｙｇｅｎ-ｉｎｄｕｃｅｄｒｅｔｉｎｏｐａｔｈｙ,ＯＩＲ）形成的影响。方法　将新生Ｃ５７ＢＬ／６Ｊ小鼠随机分为３组,分别为常氧对照组、ＯＩＲ模型组以及清除巨噬细胞的ＯＩＲ组。将后两组小鼠于生后第７天（Ｐ７）至Ｐ１２置于体积分数（７５±２）％高氧氧箱中以诱导ＯＩＲ。清除巨噬细胞的ＯＩＲ组小鼠于Ｐ９、Ｐ１１、Ｐ１３和Ｐ１５接受腹腔注射氯膦酸二钠脂质体４次以清除全身单核-巨噬细胞,ＯＩＲ模型组小鼠则于上述４个时间点接受腹腔注射ＰＢＳ脂质体。三组小鼠均于Ｐ１７时取右眼行视网膜铺片和Ｌｅｃｔｉｎ染色,观察视网膜出血及血管生长情况;取左眼行视网膜切片和ＨＥ染色,观察视网膜组织病理学变化。结果　与常氧对照组相比,ＯＩＲ模型组小鼠视网膜存在出血现象,清除巨噬细胞的ＯＩＲ组出血有所减轻。ＯＩＲ模型组小鼠视网膜血管形态呈异常增殖的团簇状,走行迂曲,而清除巨噬细胞的ＯＩＲ组小鼠视网膜新生血管异常形态减轻。与ＯＩＲ模型组相比,清除巨噬细胞的ＯＩＲ小鼠视网膜无血管区及新生血管区面积均显著减小［（１７．１９±０．５８）％、（１０．３８±０．５３）％,ｔａ＝８．６８０,Ｐ＜０．０１;（４．６０±０．１５）％、（２．５１±０．１３）％,ｔｎ＝１０．８３,Ｐ＜０．０１］,突破内界膜新生血管内皮细胞核数目明显减少（１８．５０±０．８５、７．１７±０．４８;ｔ＝１１．６６,Ｐ＜０．０１）。结论　清除巨噬细胞可减轻小鼠ＯＩＲ严重程度,提示巨噬细胞对视网膜新生血管形成具有促进作用。     

Role of unc5b in retinal neovascularization in mice with oxygen-induced retinopathy

AIM: To explore the role of unc5b in retinal neovascularization in murine oxygen-induced retinopathy (OIR). METHODS: On postnatal 7(P7), C57BL/6J mice were exposed to 75%±2% oxygen for 5 days. On postnatal 12(P12), the mice were brought back to the room air (21% oxygen) to induce retinal neovascularization. Western blot analysis was performed to examine the temporal expression of unc5b in murine retinas. Double staining for unc5b and isolectin B4 were employed to determine the location of unc5b in murine retinas. The effect of unc5b on retinal neovascularization was evaluated by intravitreal injection of unc5b-FC in mice with OIR. Retinal neovascularization was measured by counting neovascular cell nuclei above the internal limiting membrane and by angiography of flat-mounted retinas perfused with fluorescein dextran. RESULTS: Compared to age-matched normal mice, the expression of unc5b was significantly increased in retinas of OIR mice on P17 and P21. Unc5b was apparently expressed in retinal vessels of OIR while being negative in normal retinal vessels. Retinal neovascularization in eyes injected with unc5b-FC was significantly reduced. CONCLUSION: Unc5b-FC can effectively inhibit retinal neovascularization induced by OIR. It may serve as a powerful and novel therapy for ischemia-induced retinal disease.     

CCR7/p-ERK1/2/VEGF signaling promotes retinal neovascularization in a mouse model of oxygen-induced retinopathy

AIM: To investigate the role of CCR7/p-ERK1/2/VEGF signaling in the mouse model of oxygen-induced retinopathy (OIR). METHODS: Neonatal C57BL/6J mice were evenly randomized into four groups: normoxia, OIR, OIR control (treated with scramble siRNA), and OIR treated (treated with CCR7 siRNA). Normoxia group was not specially handled. Postnatal day 7 (P7) mice in the OIR group were exposed to 75%±5% oxygen for 5d (P7-P12) and then maintained under normoxic conditions for 5d (P12-P17). Mice in the OIR control and OIR treated groups were given injections of scramble or CCR7 siRNA plasmid on P12 before returning to normoxic conditions for 5d (P12-P17). Retina samples were collected from all mice on P17, stained with adenosine diphosphatase (ADPase), and retinal neovascularization (RNV) was assessed. Retinas were also stained with hematoxylin and eosin (H&E) for RNV quantitation. The distribution and expression of CCR7, p-ERK1/2 and vascular endothelial growth factor (VEGF) were assessed via immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: High oxygen promoted retinal neovascularization (P<0.05) and increased the number of endothelial nuclei in new vessels extending from the retina to the vitreous body; CCR7 promoted this process (P<0.05). CCR7 and VEGF mRNA were expressed at higher levels in the OIR and OIR control groups than in the normoxia and OIR treated groups. CCR7, p-ERK1/2, and VEGF protein were expressed in the retinas of mice in the OIR and OIR control groups. Intravitreal injection of CCR7 siRNA significantly reduced CCR7, p-ERK1/2, and VEGF expression in the OIR mouse model (all P<0.05). CCR7 significantly enhanced the neovascularization and non-perfusion areas in the OIR group (P<0.05). CCR7 siRNA significantly reduced levels of p-ERK1/2 and VEGF as compared to OIR controls (P<0.05). CONCLUSION: These results suggest that CCR7/p-ERK 1/2/VEGF signaling plays an important role in OIR. CCR7 may be a potential target for the prevention and treatment of retinopathy of prematurity.     

Inhibition of retinal angiogenesis by PEDF

AIM: To investigate the effect of PEDF on retinal neovascularization in mice. METHODS: 40 7-day-old C57BL/6J mice was exposed to 750mL/L oxygen for 5 days and then to normal situation to produce the murine model of oxygen-induced retinopathy(OIR). One eye of the mouse was regarded as experimental one and the other served as control. Eyes in experimental group received intravitreal injection of PEDF and eyes in control group received intravitreal injection of PBS at postnatal day 12. All mice were executed at postnatal day 17. The changes of retinal vessels of mice were observed by ADPase histochemical technique. The inhibitory effect of PEDF on retinal neovascularization was evaluated by counting the endotheliocyte nuclei of new vessels which extended from retina to vitreous in the tissue slice of HE staining. RESULTS: Neovascularization was reduced, retinal blood vessels distributed regularly and non-perfusion areas were not found in eyes of experimental group compared with control group. The number of endotheliocyte nuclei of new vessels extending from retina to vitreous was significantly less in the eyes of experimental group (10.18±1.74) than that in control group (38.89±2.98) (P <0.01). Retinal toxicity and inflammatory reactions were not found in tissue slice. CONCLUSION: PEDF inhibits retinal angiogenesis in OIR and the feasibility should be determined for use of PEDF in ocular angiogenesis treatment.     

靶向血管内皮生长因子的RNA干扰抑制氧诱导视网膜病变小鼠视网膜新生血管生成的研究

目的探讨玻璃体腔注射靶向血管内皮生长因子（VEGF）的RNA干扰慢病毒抑制氧诱导视网膜病变（OIR）小鼠视网膜新生血管生成的作用及其机制。方法实验研究。构建4对针对靶基冈小鼠VEGF的siRNA干扰载体,筛选并进行慢病毒包装。60只C57Bif6J小鼠分成4组（每组15只）：正常对照组．OIR模型组,OIR＋空载体组,OIR＋VEGF-RNA干扰组。OIR＋空载体组和OIR＋VEGF-RNA干扰慢病毒组的小鼠在生后第5天玻璃体腔注射相应的1μ1的7.5×10^7空载体慢病毒和VEGF-RNA干扰慢病毒。后3组小鼠在生后第7天建立OIR模型。第17天时FITC-Dextran灌注视网膜铺片观察4组小鼠视网膜血管形态及面积变化,视网膜铺片免疫荧光染色检测紧密连接蛋白Claudin-5和Occludin的分布变化．Westernblot检测VEGF、磷酸肌醇3激酶（P13K）、酪氨酸蛋白激酶SRC、磷酸化细胞外信号调节激酶（P-ERK）蛋白表达量的变化。数据采用单因素方差分析进行比较。结果FITC-Dextran灌注视网膜铺片显示正常组视网膜血管分布呈均匀网状;RNA干扰组新生血管面积（0．271399mm^2）明显较OIR模型组（1.212782mm^2）、空载体组（1．152504mm^2）少（F=449．924,P〈0．01）。OIR模型组和空载体组间差异无统计学意义,其余两两间差异均有统计学意义（P〈0．01）。视网膜铺片免疫荧光染色显示紧密连接蛋白Claudin-5和Occludin在RNA干扰组中与正常组相似,呈均匀光滑线性分布,而在OIR模型组、空载体组的分布中断、不均匀,在新生血管团中可见团块状的强荧光;VEGF的RNA干扰组中VEGF、P13K、酪氨酸蛋白激酶SRC和p-ERK的蛋白表达量较OIR模型和空载体组低。结论玻璃体腔注射靶向VEGF的RNA干扰慢病毒能有效抑制OIR小鼠模型中VEGF及其下游通路蛋白的表达．从而抑制视网膜新生血管的形成．为临床上早产儿视网膜病变的防治提供了新思路和新途径。     

转录因子Islet-1在氧诱导血管增生性视网膜病变中的表达

目的:研究高氧诱导的视网膜新生血管模型鼠中转录因子Islet-1的表达差异。方法:采用高氧诱导的方法制作鼠视网膜新生血管模型,运用荧光造影视网膜铺片及视网膜切片苏木精-伊红染色观察视网膜新生血管的形态。于小鼠出生后第7,12,14,17,26d取视网膜组织,采用Real-time PCR及Western blot技术测定视网膜组织中Islet-1的表达水平。结果:模型组视网膜铺片及组织切片可见大量视网膜新生血管形成。小鼠出生后第7d,模型组与正常组视网膜组织中Islet-1表达水平无明显差异;小鼠出生后第12~14d,模型组视网膜组织中Islet-1表达水平明显上调;出生后17d,模型组视网膜组织中Islet-1表达水平仍高于正常组;出生后26d,随着视网膜新生血管消退,视网膜组织中Islet-1表达水平降至正常水平。结论:模型鼠视网膜新生血管发生过程中,持续缺氧的视网膜组织通过增加转录因子Islet-1的表达,从而诱导视网膜新生血管的发生。     

慢病毒介导环腺苷酸反应成分结合蛋白1特异性小干扰RNA对小鼠视网膜新生血管的抑制作用

目的 观察玻璃体腔注射慢病毒介导环腺苷酸反应成分结合蛋白1（CREB1）特异性小干扰RNA（siRNA）对小鼠视网膜新生血管的抑制作用。方法 构建针对小鼠靶基因CREB1 siRNA载体,筛选并进行慢病毒包装。将140只C57BL/6J小鼠均分为正常对照组、氧诱导视网膜病变（OIR）模型组、空载体组和CREB1干扰组。正常对照组小鼠在正常空气中饲养。OIR模型组、空载体组和CREB1干扰组小鼠均于出生后7 d建立OIR模型。空载体组及CREB1干扰组小鼠于出生后5 d分别行玻璃体腔注射慢病毒 绿色荧光蛋白（GFP）空载体和CREB1-RNA干扰慢病毒载体1.0 μl。利用突破内界膜的新生血管内皮细胞核数和荧光血管造影视网膜铺片评价视网膜新生血管情况;计算小鼠视网膜新生血管和无灌注区面积;逆转录聚合酶链反应和蛋白质免疫印迹法检测小鼠视网膜CREB1 mRNA和磷酸化CREB1（P-REB1）蛋白表达,以及血管内皮生长因子（VEGF）-A、丝氨酸/苏氨酸蛋白激酶（Akt）、磷脂酰肌醇3激酶（PI3K）的mRNA和蛋白表达情况。结果 OIR模型组、空载体组突破内界膜的血管内皮细胞核计数较正常对照组明显增加,差异有统计学意义（P＜0.05）;CREB1干扰组突破内界膜的血管内皮细胞核计数较OIR模型组、空载体组明显减少,差异有统计学意义（P＜0.05）。OIR模型组、空载体组小鼠视网膜新生血管面积和无灌注区面积均较正常对照组明显增大,CREB1干扰组小鼠视网膜新生血管面积和无灌注区面积均较OIR模型组和空载体组明显缩小。4组小鼠视网膜新生血管面积和无灌注区面积比较,差异均有统计学意义（F=67.220、110.090,P＜0.05）。OIR模型组、空载体组小鼠视网膜CREB1 mRNA和P-CREB蛋白表达以及VEGF-A、Akt、PI3K mRNA和蛋白表达均较正常对照组明显增加;CREB1干扰组小鼠视网膜CREB1 mRNA和P-CREB蛋白表达以及VEGF-A、Akt、PI3K mRNA和蛋白表达较OIR模型组、空载体组明显下降。4组小鼠视网膜CREB1、VEGF-A、Akt、PI3K mRNA表达比较,差异均有统计学意义（F=6.087、5.464、6.191、8.627,P＜0.05）。4组小鼠视网膜P-CREB1、VEGF-A、Akt、PI3K 蛋白表达比较,差异也有统计学意义（F=162.944、13.861、19.710、22.827,P＜0.05）。结论 玻璃体腔注射慢病毒介导的CREB1 siRNA转染视网膜可有效抑制氧诱导小鼠视网膜新生血管的形成。     

轴突导向因子-1 mRNA在氧诱导血管增生性视网膜病变中的表达

目的研究高氧诱导的视网膜新生血管模型鼠中轴突导向因子-1（Netrin-1）mRNA的表达差异。方法采用高氧诱导的方法制作鼠视网膜新生血管模型;运用荧光造影视网膜铺片及视网膜切片苏木精-伊红染色观察视网膜新生血管的形态。于出生后第12、14、17d取小鼠视网膜,采用RT-PCR测定Netritt-1mRNA的表达水平。结果模型组视网膜铺片及组织切片可见大量视网膜新生血管形成。出生后12d,模型组与正常组视网膜组织中Netrin-1mRNA表达水平无明显差异;出生后14d,模型组视网膜组织中Netrin-1mRNA表达水平明显上调;出生后17d模型组视网膜组织中Netrin-1mRNA表达水平仍高于正常组。结论模型鼠视网膜新生血管发生过程中,持续缺氧的视网膜组织可能从转录水平增加Netrin-1的表达,从而诱导视网膜新生血管的发生。     

精氨酸-谷氨酰胺在幼鼠早产儿视网膜病变模型中的应用(英文)

目的:观察精氨酸-谷氨酰胺(Arg-Gln)对早产儿视网膜病变动物模型视网膜新生血管的抑制作用。方法:48只7日龄的C57BL/6J新生鼠暴露在750mL/L高氧环境中5d,然后回到正常空气中建立早产儿视网膜病变的动物模型。在鼠龄12d时实验组(36只)新生鼠每天两次腹腔注射Arg-Gln(剂量分别为1.0,3.0,5.0g/kg,每组12只),连续注射5d;对照组(12只)每天两次腹腔注射PBS,连续5d。所有小鼠均于17d处死,视网膜铺片,ADP酶染色观察视网膜血管情况。HE染色,在光学显微镜下观察并计数突破视网膜内界膜的血管内皮细胞细胞核数目。Real-time RT-PCR方法测量每组视网膜VEGF mRNA水平。结果:与对照组相比,实验组以剂量依赖方式无灌注区面积和新生血管团逐渐减少;实验组中最大剂量组[5.0g/(kg·d)]突破内界膜的内皮细胞细胞核数目比对照组大约减少75%(P<0.01);实验组视网膜VEGF mRNA水平与对照组相比明显下降。结论:Arg-Gln能够有效抑制早产儿视网膜病变动物模型视网膜新生血管的生成,可能为临床提供一种预防和治疗早产儿视网膜病变安全有效的新方法。     

Activated NAD(P)H oxidase from supplemental oxygen induces neovascularization independent of VEGF in retinopathy of prematurity model

PURPOSE: To study NAD(P)H oxidase-dependent outcomes after oxygen stresses that are similar to those experienced by preterm infants today using a rat model of retinopathy of prematurity. METHODS: Within 4 hours of birth, pups and their mothers were cycled between 50% and 10% oxygen daily for 14 days and were returned to room air (21% O2, 50/10 oxygen-induced retinopathy [OIR]) or supplemental oxygen (28% O2, 50/10 OIR+SO) for 4 days. Pups received intraperitoneal injections of the specific NAD(P)H oxidase inhibitor apocynin (10 mg/kg/d) or of PBS from postnatal day (P)12 to P17, and some received intraperitoneal injections of hypoxyprobe before kill. Intravitreous neovascularization (IVNV), avascular/total retinal areas, vascular endothelial growth factor (VEGF), NAD(P)H oxidase activity, or hypoxic retina (conjugated hypoxyprobe) were determined in neurosensory retinas. Human retinal microvascular endothelial cells (RMVECs) treated with apocynin or control were exposed to 1% or 21% O2 and assayed for phosphorylated (p-)Janus kinase (JNK) and NAD(P)H oxidase activity. RESULTS: Retinas from 50/10 OIR+SO had increased NAD(P)H oxidase activity and lower VEGF than did retinas from 50/10 OIR. Apocynin treatment reduced the IVNV area and hypoxic retina in 50/10 OIR+SO. RMVECs treated with 1% O2 had increased p-JNK compared with RMVECs exposed to room air. CONCLUSIONS: Different oxygen stresses activate NAD(P)H oxidase to varying degrees to trigger disparate pathways (angiogenesis or apoptosis). The oxygen stresses and outcomes used in this study are relevant to human ROP and may explain some of the complexity in the pathophysiology of ROP resulting from oxygen exposure.     

Toll样受体4对氧诱导视网膜新生血管发生的促进作用及其机制

背景 研究发现,Toll样受体(TLRs)在视网膜新生血管的形成中发挥重要作用,但其作用机制尚不清楚. 目的 探讨TLR4对小鼠氧诱导视网膜病变(OIR)中新生血管生成的作用及其机制. 方法 将18只7日龄新生C57BL/6J小鼠以随机数字表法按小鼠窝别分为常氧组、OIR组及OIR+脂多糖(LPS)-EB组,常氧组小鼠在正常氧环境中饲养,OIR组及OIR+LPS-EB组小鼠于生后第7天(P7)与母鼠置于体积分数(75±2)％高氧氧箱中饲养5d以诱导OIR模型,于P12在正常氧环境中饲养,OIR+LPS-EB组P12小鼠腹腔内注射LPS-EB,剂量为1 mg/kg,OIR组同法注射PBS.各组摘取P17小鼠眼球于质量分数4％多聚甲醛中固定并行视网膜铺片和Lectin染色,计算视网膜无血管区和新生血管区面积占全视网膜的百分比.各组制备P17小鼠眼后节组织冰冻切片并行免疫荧光检测,计数小鼠5 mm视网膜全长活化小胶质细胞数目;采用CD11b和TLR4免疫荧光双标记法检测各组小鼠视网膜小胶质细胞中CD11b、TLR4的表达及其分泌血管内皮生长因子(VEGF)、白细胞介素1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的水平.结果 常氧组P17小鼠视网膜血管分布和形态正常,OIR组和OIR+LPS-EB组P17小鼠视网膜中央均出现无血管区和新生血管簇,OIR+LPS-EB组小鼠视网膜无血管区面积比为(18.47±1.32)％,新生血管面积比为(3.29±0.85)％,分别大于OIR组的(15.78±1.44)％和(1.77±0.19)％,差异均有统计学意义(t=3.36,P=0.01;t=4.22,P=0.00).免疫荧光结果显示,常氧组P17小鼠视网膜中活化小胶质细胞数量极少,OIR组和OIR+LPS-EB组P17小鼠视网膜中活化小胶质细胞荧光强度增强,活化小胶质细胞数量增加,其中OIR+LPS-EB组小鼠视网膜中小胶质细胞数明显多于OIR组,差异有统计学意义[(95.50士4.77)/5 mm与(74.83±4.17)/5 mm,t=8.00,P＜0.01].常氧组小鼠视网膜中TLR4阳性小胶质细胞数量极少,OIR组和OIR+LPS-EB组小鼠视网膜中TLR4荧光增强,TLR4阳性小胶质细胞数量明显增加,OIR+LPS-EB组小鼠视网膜中TLR4阳性小胶质细胞数目明显多于OIR组,差异有统计学意义[(49.50±6.38)/5 mm与(28.17±6.24)/5 mm,t=5.86,P＜0.01)].常氧组P17小鼠视网膜中CD11b和VEGF/IL-1β/TNF-α共表达的小胶质细胞数量分别为(1.17±0.75)/5 mm、0和0,而OIR+LPS-EB组小鼠视网膜中共表达CD11b和VEGF/IL-1β/TNF-α的小胶质细胞数量多于OIR组,差异有统计学意义[(44.50±8.78)/5mm与(28.50±5.61)/5 mm,F=44.07,P＜0.01;(24.10±6.49)/5 mm与(16.00±3.46)/5 mm,F=11.31,P＜0.01;(33.83±14.82)/5 mm与(23.00±2.83)/5 mm,t=19.92,P＜0.01]. 结论 TLR4可促进OIR小鼠视网膜新生血管的生成,其作用机制可能与TLR4激活视网膜小胶质细胞中相关的下游信号通路,促进血管生长因子及炎性因子的释放有关.