脂联素对小鼠氧诱导视网膜病变的保护作用

目的：观察脂联素对C57BL/6J小鼠氧诱导视网膜病变的影响。

方法：将新生C57BL/6J小鼠随机分为3组,分别为常氧对照组、生理盐水注射氧诱导视网膜病变(oxygen-induced retinopathy,OIR)组和脂联素注射OIR组。将后两组小鼠于生后第7d(P7)至P12置于体积分数75%±2%高氧氧箱中以诱导OIR模型。脂联素注射OIR模型组在P7～P15每天接受腹腔注射重组鼠脂联素蛋白(3.0μg/g),生理盐水注射OIR组则于上述相同时间点注射同等剂量的生理盐水。三组小鼠均于P17时取右眼行视网膜铺片和Lectin染色,观察视网膜中央无血管区及病理性新生血管的情况; 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; 应用Western-blot测定左眼视网膜组织中iNOS、nNOS、eNOS的表达; 取右眼视网膜组织定量检测ROS/RNS含量以及NO水平。

结果：脂联素注射OIR组小鼠视网膜中央无血管区面积较生理盐水注射OIR组明显减少(t=7.304,P<0.01),病理性新生血管数目明显减少(t=2.654,P<0.01); 脂联素注射OIR组小鼠视网膜组织ROS含量较生理盐水注射组明显降低(t=13.349,P<0.01); 脂联素注射OIR组小鼠视网膜组织NO含量明显高于生理盐水注射组(t=3.023,P<0.01),iNOS表达明显低于生理盐水注射组(t=5.112,P<0.01),eNOS表达明显高于生理盐水注射组(t=7.421,P<0.01),nNOS的表达无统计学差异(t=1.074,P>0.01)。

结论：脂联素可以通过激活内源性eNOS促进生理性NO生成,同时又能抑制ROS/RNS的产生,在OIR进程中发挥视网膜血管的保护作用。     

PEDF对氧诱导视网膜病变小鼠视网膜MCP-1表达的影响

目的：观察色素上皮衍生因子(pigment epithelium-derived factor，PEDF)在氧诱导视网膜病变(oxygen-induced retinopathy，OIR)中对小鼠视网膜新生血管(retinal neovascularization，RNV)和单核细胞趋化因子-1(monocyte chemoattractant protein-1，MCP-1)表达的影响，探讨PEDF对缺血缺氧性视网膜病变的保护作用和机制。

方法：取7日龄C57BL/6J新生小鼠160只，将120只7日龄小鼠与哺乳母鼠共同置于氧浓度为(75±2)%的氧环境内饲养5d，然后返回正常氧环境中饲养5d，建立OIR模型； 40只小鼠始终置于正常氧环境饲养。分别于12日龄和14日龄给予PEDF药物治疗组小鼠右眼玻璃体腔注射PEDF(2μg/μL)各1μL，给予PBS治疗对照组和正常对照组小鼠右眼玻璃体腔注射等量的磷酸盐缓冲液(phosphate buffered saline，PBS)。所有小鼠于17日龄麻醉处死后取视网膜，采用视网膜铺片和Lectin染色法观察各组小鼠病理性新生血管的生成情况； Western-blot检测PEDF和MCP-1蛋白在各组小鼠视网膜的表达； 实时荧光定量逆转录多聚酶链反应(RT-PCR)检测各组小鼠视网膜PEDF和MCP-1 mRNA的表达。

结果：视网膜铺片和Lectin染色结果显示OIR模型组RNV面积较正常组显著增大，差异有统计意义(P<0.01)，PEDF药物治疗组RNV面积较PBS治疗对照组明显减小，差异有统计意义(P<0.01)。Western-blot和RT-PCR结果显示，OIR模型组MCP-1蛋白和mRNA的表达水平均明显高于正常组，差异有统计意义(均P<0.05)； OIR模型组PEDF蛋白和mRNA的表达水平均明显低于正常组，差异有统计意义(均P<0.01)； PEDF药物治疗组MCP-1蛋白和mRNA的表达量较PBS治疗对照组均显著减少，差异有统计意义(均P<0.05)； PEDF药物治疗组MCP-1蛋白和mRNA的表达量较正常对照组升高，但差异均无统计学意义(均P>0.05)。

结论：PEDF能够抑制OIR小鼠视网膜新生血管形成，同时下调MCP-1在OIR小鼠视网膜的表达，后者可能是其抑制新生血管形成从而发挥视网膜保护作用的机制之一。     

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.     

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.     

血管内皮生长因子干扰RNA对鼠视网膜中血管内皮生长因子mRNA表达的抑制作用

目的 研究血管内皮生长因子（vascular endothelial cell growth factor,VEGF）小片段干扰RNA（small interference RNA,siRNA）对鼠视网膜VEGF mRNA的抑制作用,探讨其对视网膜新生血管治疗的可行性.方法 体外培养人鼻咽癌细胞（CNE-2Z）,分成正常氧培养组（20% O2）和低氧培养组（1% O2）.采用脂质体（LF 2000）将VEGF siRNA转染两组细胞,RT-PCR检测VEGF mRNA的表达,确立VEGF siRNA对VEGFmRNA的抑制效率.然后,建立高浓度氧（75%）诱导的C57BL./6J小鼠视网膜新生血管动物模型,以脂质体为载体,将VEGF siRNA重组质粒注射到鼠玻璃体腔内,RT-PCR检测视网膜组织中VEGF mRNA的表达水平.结果 正常氧培养的CNE-2Z细胞有VEGF mRNA表达,低氧状态下VEGFmRNA表达增多,两者之间差异有显著性（P＜0.01）;与未转染组和转染空载体组相比,在正常氧和低氧状态下,VEGFsiRNA均能明显抑制VEGFmRNA的表达（P＜0.01）;正常氧状态下VEGF siRNA的抑制效率比低氧状态高.高浓度氧诱导的C57BL/6J小鼠视网膜新生血管动物模型中,玻璃体腔注射VEGF siRNA组视网膜组织中VEGF mRNA表达明显下降（P＜0.01）.结论 VEGF特异的siRNA能有效地抑制人鼻咽癌细胞CNE-2Z和C57BL/6J 小鼠视网膜新生血管动物模型视网膜中VEGF mRNA的表达.     

抗血管内皮生长因子对氧诱导视网膜新生血管病变模型小鼠视网膜多巴胺含量的影响

目的　研究抗血管内皮生长因子（VEGF）融合蛋白康柏西普（Conbercept）玻璃体内注射对小鼠氧诱导视网膜新生血管病变（OIR）模型中视网膜多巴胺（DA）含量的影响。方法　普通级7 d龄C57BL/6小鼠100只,随机分为空白对照组、OIR组、OIR+生理盐水（NS）组及OIR+ Conbercept组,每组25只。其中空白对照组小鼠在常氧环境中饲养至17 d龄。OIR组、OIR+NS组及OIR+ Conbercept组小鼠通过高流量吸氧建立OIR模型,并在此环境中饲养至12 d龄,OIR+NS组和OIR+Conbercept组小鼠分别行右眼玻璃体内注射1 μL NS、1 μL Conbercept后,在常氧环境中饲养至17 d龄,处死。取各组小鼠右眼眼球行HE染色及视网膜铺片,观察突破内界膜的视网膜血管内皮细胞核数及血管分布;行Western blot检测各组小鼠视网膜中酪氨酸羟化酶（TH）、血管内皮生长因子（VEGF）表达量;行高效液相色谱仪检测各组小鼠视网膜DA含量。结果　空白对照组小鼠视网膜各层清晰,未见明显无灌注区及新生血管。与空白对照组相比,OIR组小鼠视网膜各层排列紊乱,视盘周围可见大片无灌注区,突破内界膜的血管内皮细胞核数、VEGF相对表达量均增加,TH相对表达量、DA含量均减少,差异均有统计学意义（均为P<0.05）;OIR+NS组与OIR组相比,小鼠视网膜中突破内界膜的血管内皮细胞核数、DA含量及VEGF、TH相对表达量差异均无统计学意义（均为P>0.05）;与OIR组及OIR+NS组相比,OIR+ Conbercept组小鼠视网膜各层排列较清晰,视盘周围可见小片状无灌注区,突破内界膜的内皮细胞核数、DA含量及VEGF、TH相对表达量均减少,差异均有统计学意义（均为P<0.05）。结论　OIR模型中视网膜DA含量及TH相对表达量均降低;玻璃体内注射Conbercept后,视网膜新生血管及VEGF相对表达量均减少,TH相对表达量、DA含量均进一步降低。     

VEGF siRNA抑制鼠视网膜新生血管的实验研究

背景血管内皮生长因子（VEGF）在视网膜新生血管的发生过程中发挥重要作用,抑制VEGF是目前视网膜新生血管治疗和预防研究的热点。VEGF小片段干扰RNA（VEGFsiRNA）在抗肿瘤新生血管的研究中已经取得了显著疗效,但对于视网膜新生血管的干预作用报道较少。目的研究VEGF siRNA对鼠视网膜新生血管的抑制作用。方法48只新生C57BL／6J幼鼠采用随机数字表法随机分为正常对照组、模型对照组、空载体组和VEGF siRNA质粒转染组,每组12只幼鼠。7日龄C57BL／6J幼鼠36只及其母鼠置于密闭的氧舱5d建立缺氧性新生血管模型,其中12只幼鼠不进行质粒转染作为模型对照组,其余24只鼠玻璃体腔内注射脂质体（LF2000）包裹的空载体质粒或VEGF siRNA表达质粒。待小鼠19日龄时获取小鼠眼球并分离视网膜,用苏木精一伊红染色法计数各组小鼠视网膜新生血管内皮细胞核的数目,用实时荧光定量聚合酶链反应（tea-time PCR）法检测视网膜中VEGF mRNA的表达,并应用免疫荧光技术检测小鼠视网膜中VEGF蛋白的表达。结果正常对照组、模型对照组、空载体组和VEGFsiRNA质粒转染组19日龄小鼠视网膜突破内界膜的内皮细胞细胞核数目分别为（0．19±0．09）个、（24．89±2．03）个、（23．65±2．15）个和（8．83±1．12）个,表明VEGFsiRNA质粒转染组小鼠的新生血管内皮细胞数明显低于模型对照组和空载体组,差异均有统计学意义（q=5．67、q=4．97,P〈0．01）。Real-time PCR检测表明,正常对照组小鼠视网膜中仅见弱的VEGF mRNA表达,而模型对照组与空载体组VEGF mRNA表达量为正常对照组的52．3倍和36．7倍,VEGF siRNA质粒转染组小鼠视网膜VEGF mRNA的表达量为正常对照组的3．5倍,明显低于模型对照组与空载体组。VEGF siRNA对VEGF mRNA的抑制率为43．39％。免疫荧光染色显示,正常对照组小鼠VEGF蛋白呈弱阳性表达,模型对照组和空载体组VEGF小鼠视网膜中VEGF蛋白表达呈强阳性,VEGF siRNA质粒转染组VEGF蛋白表达明显减弱。结论玻璃体腔注射VEGF siRNA表达质粒可有效抑制C57BL／6J小鼠氧诱导视网膜病变模型新生血管的形成。     

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。     

Caspase-1对氧诱导视网膜病变中小胶质细胞参与视网膜新生血管生成的促进作用及其机制

目的：探讨Caspase-1对小鼠氧诱导视网膜病变(oxygen-induced retinopathy,OIR)中小胶质细胞参与视网膜新生血管生成的作用及其机制。

方法：随机将12只7日龄(P7)C57BL/6J小鼠分为正常组、OIR组和OIR+VX-765组,正常组在正常氧环境中饲养,其余两组构建OIR模型; P12～P16,OIR+VX-765组和OIR组分别每天腹腔注射Caspase-1抑制剂VX-765(4mg/kg)和等量0.4%聚乙二醇(VX-765溶剂); 于P17制作视网膜铺片行Lectin染色,比较三组间视网膜无血管区和新生血管区面积的大小; 采用免疫荧光染色法观察视网膜组织中Caspase-1的表达和活化小胶质细胞的分布。培养小胶质细胞BV-2细胞分为对照组、缺氧组及抑制剂组,抑制剂组和缺氧组分别经VX-765和0.4%聚乙二醇预处理3h后,缺氧条件下培养24h; 对照组常规培养相同时间。通过Western blot检测Caspase-1、p20(Caspase-1活化形式)、IL-1β和VEGF的蛋白表达变化; 用各组BV-2细胞培养上清液作为条件培养基,刺激培养血管内皮细胞RF/6A,进行管腔形成和细胞迁移实验,并比较各组间的差异。

结果：P17正常组小鼠视网膜血管化完全,未见明显无血管区及视网膜新生血管; OIR组视网膜无血管区和新生血管面积百分比分别为16.58%±1.14%、4.00%±0.41%; OIR+VX-765组两者明显减少,分别为12.23%±1.02%和2.16%±0.52%(P<0.01)。免疫荧光染色结果显示,Caspase-1在正常小鼠视网膜组织中表达较弱,在OIR小鼠中主要在神经节细胞层和内丛状层有明显的阳性表达,并与活化的小胶质细胞有明确的共定位。Western blot检测结果显示,缺氧处理的培养小胶质细胞BV-2中Caspase-1、p20、IL-1β和VEGF蛋白表达明显提高,而Caspase-1抑制剂则可明显下调p20、IL-1β和VEGF的蛋白表达水平(P<0.05)。管腔形成和细胞迁移实验结果显示,RF/6A细胞经缺氧组BV-2培养上清液处理后,管腔形成长度和细胞迁移数目分别为271±12和347±34个,而加入抑制剂后,二者明显减少,分别为171±22和212±27个(P<0.05)。

结论：在小鼠OIR中,Caspase-1能够调节小胶质细胞促进视网膜新生血管的生成,其作用机制可能与Caspase-1活化小胶质细胞中其下游炎性效应分子IL-1β,并释放VEGF相关。     

Delta样配体4单克隆抗体对视网膜新生血管形成和血管内皮生长因子表达的影响

背景 Delta样配体4(Dll4)参与视网膜内细胞的发育和血管的发生过程,并与血管内皮生长因子(VEGF)共同参与诱导和挑选尖端细胞的过程.Dll4在视网膜新生血管形成中的作用及其与VEGF表达的关系值得关注. 目的 研究Dll4单克隆抗体玻璃体腔内注射后对视网膜中Dll4、VEGF及其受体表达和视网膜新生血管形成的影响.方法 将5日龄的新生SPF级SD大鼠与母鼠一起在含体积分数为(80±2)％氧气的密闭玻璃箱内饲养至12日龄,然后返回自然环境下饲养至17日龄以建立大鼠氧诱导视网膜病变(OIR)模型.于模型大鼠12日龄时右眼玻璃体腔内注射Dll4单克隆抗体2.5 μl(0.5 μg)为Dll4单克隆抗体注射组,左眼以同样的方式注射等体积PBS作为PBS对照组.于大鼠17日龄时处死大鼠并分离视网膜.应用逆转录PCR(RT-PCR)法检测两组大鼠视网膜中Dll4、VEGF、VEGF受体-1(VEGFR-1)、VEGFR-2、神经纤维网蛋白-1(neuropilin-1)mRNA的表达情况,采用视网膜铺片ADP酶染色法观察大鼠视网膜新生血管形态,采用视网膜切片苏木精-伊红染色法计数突破内界膜的血管内皮细胞核数目,评估新生血管的严重程度.两组间各检测指标的差异比较采用配对t检验. 结果 Dll4单克隆抗体注射组大鼠视网膜内Dll4 mRNA表达灰度值(Dll4 mRNA/β-actin mRNA)为0.22±0.06,明显低于PBS对照组的0.98±0.13,差异有统计学意义(t=21.839,P=0.000),而2个组间VEGF mRNA及其受体VEGFR-1 mRNA、VEGFR-2 mRNA表达灰度值的差异均无统计学意义(t=0.463,P=0.649;t=1.687,P=0.109;t=-1.674,P=0.111);与PBS对照组比较,Dll4单克隆抗体注射组小鼠视网膜中neuropilin-1 mRNA的表达量明显升高,差异有统计学意义(0.73±0.08 vs.0.64±0.07)(t=-2.677,P=0.015).Dll4单克隆抗体注射组大鼠视网膜新生血管密度明显高于PBS对照组.Dll4单克隆抗体注射组大鼠视网膜突破内界膜的血管内皮细胞数为(63.6±11.6)个/张,PBS对照组为(35.1±5.2)个/张,差异有统计学意义(t=-7.879,P=0.000). 结论 Dll4在视网膜新生血管形成的过程中发挥重要作用,并可能通过对VEGFR的反馈抑制发挥抑制病理性新生血管过度形成的作用.     

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。     

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.     

Intravitreous high expression level of netrin-1 in patients with proliferative diabetic retinopathy

 Abstract Purpose: To investigate the significance of netrin-1 and vascular endothelial growth factor (VEGF) in the pathogenesis of retinal angiogenesis, the levels of netrin-1 and VEGF in the vitreous fluid and serum of the proliferative diabetic retinopathy (PDR) and non-proliferative diabetic retinopathy (non-PDR) patients were measured. We then determined the netrin-1 and VEGF expression in the oxygen induced retinopathy (OIR) mice retina. Methods: A total of 18 eyes from 18 patients were included in our study and 10 of them were collected from PDR patients and 8 from non-PDR patients. Undiluted vitreous fluid samples were collected during pars plana vitrectomy. Appropriate blood samples were collected if possible. Netrin-1 and VEGF levels in the vitreous fluid and plasma were determined by Enzyme-linked Immunosorbent Assays. OIR mice models were established, and netrin-1 and VEGF levels were determined by immunohistochemistry analysis.  Results: The levels of netrin-1 and VEGF in the vitreous of PDR patients were significantly higher than those in the controls (Mediannetrin-1=509.94 vs. 85.91 pg/ml, P<0.001 and Median VEGF=762.60 vs. 77.52 pg pg/ml, P<0.001). Netrin-1 was mainly expressed in GCL and INL of the retina in mice. Both netrin-1 and VEGF were up-regulated in OIR mice. Conclusion: Netrin-1 and VEGF levels were elevated in vitreous fluid of the PDR patients and the OIR mice retina. Therefore, netrin-1 may play an important role in pathological retinal angiogenesis.     

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.     

PEDF抑制鼠视网膜新生血管的实验研究(英文)

目的:观察PEDF对氧诱导的血管增生性视网膜病变动物模型视网膜新生血管的抑制作用。方法:40只7日龄的C57BL/6J新生鼠暴露在750mL/L高氧环境中,然后回到正常空气中建立氧诱导的血管增生性视网膜病变的动物模型。随机取1眼为实验眼,在出氧箱时(12d)玻璃体腔注射PEDF,另1眼为对照组,玻璃体腔注射PBS。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。结果:与对照组相比,实验组视网膜血管分布规则,未见明显的无灌注区形成;实验组突破内界膜的内皮细胞细胞核数目(10.18±1.74)比对照组(38.89±2.98)明显减少(P<0.01) ;组织切片未见视网膜毒性及炎症反应。结论:PEDF能够有效抑制视网膜新生血管的生成。     

单核细胞趋化蛋白-1在氧诱导视网膜病变新生小鼠模型中的表达

背景 单核细胞趋化蛋白-1( MCP-1)是趋化因子家族中的主要成员之一,在肿瘤、炎症和糖尿病视网膜病变(DR)等新生血管性疾病中起着重要的作用,但关于MCP-1在氧诱导视网膜病变(OIR)发生过程中的表达及其作用的研究鲜有报道. 目的 观察OIR小鼠视网膜中MCP-1的表达,探讨MCP-1在视网膜血管发育和视网膜新生血管形成过程中的作用. 方法 SPF级C57BL/6J小鼠120只,按随机数字表法随机分为OIR组和正常对照组,每组60只.OIR组将出生后7d的新生小鼠在体积分数75％氧环境下饲养5d,然后返回正常大气环境中;正常对照组小鼠始终置于正常大气环境中饲养.OIR组和正常对照组分别在出生后5、7、12、14、17、21 d随机抽取10只小鼠,摘除右眼球,采用免疫组织化学法检测MCP-1蛋白在小鼠视网膜中的表达情况,采用逆转录-聚合酶链反应(RT-PCR)法检测MCP-1 mRNA在小鼠视网膜中的表达. 结果 正常对照组小鼠在出生后的第5天即有MCP-1在视网膜的内核层和节细胞层表达,12d时表达MCP-1的阳性细胞数达到高峰,其他日龄时呈基线表达.OIR组12d时表达MCP-1的阳性细胞数轻度增多,14d时阳性细胞数显著增多,之后迅速下降至正常水平.正常对照组在5d时可检测到MCP-1 mRNA表达,12d时显著上调,之后随小鼠日龄的增加,MCP-1 mRNA表达迅速下降,14、17、21 d表达基本呈基线水平.OIR组12 d时,MCP-1 mRNA较正常对照组下降,在新生血管形成关键期,即14 d时表达显著上调,之后迅速下降至正常水平.OIR组和正常对照组间比较采用完全随机分组的两因素方差分析,F分组=5.230,P=0.028;F日龄=6.898,P=0.001.结论 小鼠视网膜发育过程始终伴随着MCP-1的表达,MCP-1的表达上调可能与小鼠视网膜血管发育和OIR模型中视网膜新生血管的形成密切相关.     

Matrix metalloproteinase-9 and vascular endothelial growth factor expression change in experimental retinal neovascularization

AIM: To investigate the signal transduction mechanism of matrix metalloproteinase-9 (MMP-9) mediated- vascular endothelial growth factor (VEGF) expression and retinal neovascularization (RNV) in oxygen-induced retinopathy (OIR) model. METHODS: C57BL/6J mice were divided into four groups: control group, OIR group, OIR control group (phosphate-buffered saline by intravitreal injection) and treated group [tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) by intravitreal injection]. OIR model was established in C57BL/6J mice exposed to 75%±2% oxygen for 5d. mRNA level and protein expression of MMP-9, TIMP-1 and VEGF were measured by real-time polymerase chain reaction and Western blotting, and located by immunohistochemistry. RESULTS: Levels of MMP-9 and VEGF in retina were significantly increased in animals with OIR and OIR control group. Levels of TIMP-1 in retina was significantly reduced in animals with OIR and OIR control group. Furthermore, a significant correlation was found between MMP-9 and VEGF. Intravitreal injection of TIMP-1 significantly reduced MMP-9 and VEGF expression of the OIR mouse model (all P<0.05). CONCLUSION: These results demonstrate that MMP-9-mediated up-regulation of VEGF promotes RNV in retinopathy of prematurity (ROP). TIMP-1 may be a potential target for the prevention and treatment of ROP.     

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.     

Efficacy of intravitreal captopril on oxygen-induced retinopathy in mice

AIM: To study the inhibitory effect of intravitreal captopril on oxygen-induced retinopathy (OIR) in mice. METHODS: Eighty postnatal day (P)7 C57BL/6J mice were randomly divided into treated group and control group with forty mice in each group. The mice were exposed to 75% ± 2% oxygen for 5 days (P7-P11) and then returned to room air for 5 days (P12-P17) to induce retinal neovascularization (RNV). Beginning on P12, the mice in treated group received daily intravitreal injections of captopril (3.0mL/kg), while those in control group received daily intravitreal injections of phosphate-buffered saline (PBS) (3.0mL/kg) through P17. After anesthetized at P17, one eye was chosen randomly as experimental eye and were enucleated. RNV was examined by Adenosine diphosphate-ase (ADPase) stained retina flat-mounts and was quantitated histologically by counting the neovascular endothelial cell nuclei anterior to inner limiting membrane (ILM). The expressions of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) were measured by immunohistochemical method. RESULTS: Comparing with control group, more regular distributions, better branch and reduced density of RNV were observed in eyes of treated group. The number of neovascular cell nuclei was less in treated group than that in control group (t=6.135, P<0.01). Stain of MMP-2 and VEGF was weaker in treated group than that in control group. CONCLUSION: The results indicate that captopril can significantly inhibit RNV in OIR mice.