抗血管内皮生长因子单克隆抗体Bevacizumab预防非肥胖糖尿病小鼠视网膜微血管增生

目的 观察抗血管内皮生长因子单克隆抗体Bevacizumab眼内注射对非肥胖糖尿病小鼠视网膜微血管增生的预防作用。 方法 选取30只非肥胖糖尿病小鼠，左眼为实验眼，右眼为对照眼。实验眼眼内注入1 μl Bevacizumab(25 mg/1 ml)溶液， 对照眼眼内注入等量生理盐水。分别在注射后1周，1、2个月时随机各选取10只鼠，取出双侧眼球，行视网膜微血管内皮细胞超微结构观察以及视网膜CD34和血管内皮生长因子（VEGF）免疫组织化学测定，计算机图像分析对比两组间阳性染色密度的差异。 结果 VEGF和CD34阳性表达均为棕黄色着色，CD34的染色定位在血管内皮细胞上。在注射后1周、1个月时，两组间VEGF表达比较，差异有统计学意义（t=21.6, t=13.5; P＜0.01 ）；注射后2个月时，两组间VEGF表达比较，差异无统计学意义（t=0.9, P＞0.05）。注射后1周时，两组间CD34表达比较，差异无统计学意义（t=1.3, P＞0.05）；注射后1、2个月时，两组间CD34表达比较，差异有统计学意义（t= 3.2, P＜0.01; t=2.7, P＜0.05）。注射后各时间段，视网膜血管内皮细胞的微观结构都未发生明显改变。 结论 Bevacizumab眼内注射可预防非肥胖糖尿病小鼠视网膜微血管的异常增生。 （中华眼底病杂志，2008，24：180-183）     

氧诱导视网膜病变鼠模型血管内皮 生长因子mRNA的表达

目的分析氧诱导视网膜病变动物模型血管内皮生长因子（VEGF）基因的调节规律，阐明早产儿视网膜病变（ROP）新生血管形成的可能机制。方法将36只7 d 龄C₅₇BL/6J幼鼠暴露在（75±2）% 浓度的高氧状态下5 d，随后在正常氧环境下5 d，作为氧诱导模型组；另24只同日龄幼鼠作为正常对照组。采用荧光素血管灌注及视网膜铺片法观察视网膜血管形态；半定量逆转录-聚合酶链反应(RP-PCR)观察各组VEGF mRNA的变化。结果氧诱导模型的视网膜血管形态特征为高氧状态下表层和深层血管的中心区出现无灌注，相对低氧状态下2 d后开始出现新生血管，其部位在中周部。RF-PCR结果显示，VEGF的表达与眼内新生血管的发生存在明确的时空对应关系，即高氧状态下，VEGF mRNA转录下降，相对低氧状态下，VEGF mRNA过度转录。结论缺氧是视网膜新生血管发生的主要原因；高氧之后的相对低氧使VEGF表达增加，可能会降低ROP新生血管的发生。(中华眼底病杂志,2005,21:292-295)     

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。     

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.     

重组血管生成抑制因子k4k5抑制视网膜新生血管形成的实验研究

目的 观察重组血管生成抑制因子k4k5 (r-k4k5)对视网膜新生血管形成的抑制作用。 方法 将鼠龄为7 d的88只C 57BL/6J幼鼠置于75%浓度的氧环境中连续生活5 d，建立氧诱导的血管增生性视网膜病变幼鼠模型。幼鼠玻璃体腔内分别注射r-k4k5 500 ng(大剂量治疗组)、250 ng(小剂量治疗组) ，对侧眼注射相同体积的平衡盐溶液(balanced salt solution，BSS)作为对照。二磷酸腺苷(adenosine diphosphate,ADP)酶染色法视网膜铺片，了解视网膜血管改变 ；用组织切片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，观察r-k4k5对视网膜新生血管形成的抑制情况。 结果 与对照组相比，治疗组视网膜血管分布规则、密度减少；治疗组突破内界膜的内皮细胞细胞核数目明显减少(P<0.001)；而且与小剂量治疗组相比较，大剂量治疗组内皮细胞细胞核数目减少，两组差异有显著性的意义(P<0.001)。组织切片未见视网膜毒性及炎症反应。 结论 r-k4k5可以抑制视网膜新生血管形成，有望成为防治血管增生性视网膜病变的一种有效的方法。 （中华眼底病杂志，2003，19：121-124）     

Bevacizumab抑制高氧诱导新生小鼠视网膜新生血管的实验研究

目的 探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)的单克隆抗体Bevacizumab(Avastin)对视网膜新生血管的抑制作用.方法 80只7 d龄清洁级C57BL/6小鼠随机分为4组,即空白对照组、高氧实验组、小剂量实验组和大剂量实验组,建立高氧诱导新生小鼠产生视网膜新生血管的动物模型,分别对小剂量实验组和大剂量实验组行玻璃体注射Bevacizumab(25 g·L-1)0.5μL、1.0μL,免疫组织化学染色法观察VEGF在视网膜各层的表达,应用CD31计算突破视网膜内界膜的新生血管内皮细胞核数,评价该药对视网膜新生血管的抑制作用.电镜观察该药对视网膜超微结构的影响.结果 VEGF在各组视网膜中均有表达,在高氧组表达明显增加,两给药组表达相对较弱,空白对照组最弱.空白对照组、高氧实验组、小剂量实验组及大剂量实验组突破视网膜内界膜的新生血管内皮细胞核数分别为(0.20±0.42)个、(16.80±6.05)个、(3.90±1.52)个、(5.10±2.88)个,统计学处理结果表明两给药组分别与高氧实验组比较差异均有显著统计学意义(P均<0.01),而两给药组之间差异无统计学意义(P>0.05).透射电镜检查显示两给药组视网膜超微结构与高氧实验组相比损伤较轻.结论 Bevacizumab能有效抑制视网膜新生血管的形成,并在一定程度上对缺氧造成的视网膜超微结构的损伤具有预防作用.     

白藜芦醇对氧诱导鼠视网膜B细胞白血病蛋白2和血管内皮生长因子表达的影响

目的探讨白藜芦醇对高氧诱导的早产儿视网膜病变新生鼠视网膜B细胞白血病蛋白2（B-cell leukemia/lymphoma-2,bcl-2）和血管内皮生长因子（vascular endothelial growth factor,VEGF）表达的影响。方法选取鼠龄为7d的Sprague-Dawley（SD）幼鼠60只,分A组（正常对照组）、B组（给氧组）和C（实验组1）、D（实验组2）、E（实验组3）三个实验组,每组12只。A组幼鼠生活在正常氧环境中,B组、C组、D组和E组幼鼠置于氧分压为（75±2）%的氧环境中连续生活5d,建立高氧诱导的早产儿视网膜病变动物模型。C、D、E三个实验组分别予白藜芦醇10mg/（kg·d）、30mg/（kg·d）、60mg/（kg·d）灌胃给药,每天一次,连续5d,所有幼鼠均于17d鼠龄处死。应用Western-blot免疫印迹法分析检测鼠视网膜bcl-2的表达,并以计算机图像分析仪进行分析;用ELISA方法检测鼠视网膜VEGF的表达。结果与A组（正常对照组）比较,B组（给氧组）视网膜bcl-2和VEGF的表达是A组的4倍,两组差异有统计学意义（P＆lt;0.01）;与B组比较,C、D、E三个实验组视网膜bcl-2和VEGF的表达逐渐减弱,呈剂量依赖关系,不同浓度的白藜芦醇实验组之间存在显著性的差异（P〈0.01）,三种不同浓度（10mg/kg、30mg/kg、60mg/kg）的白藜芦醇均显著抑制视网膜bcl-2和VEGF的表达,其抑制率分别为11.09%、38.05%、69.76%和3.42%、23.04%、43.69%。结论白藜芦醇可显著抑制高氧诱导的早产儿视网膜病变新生鼠视网膜bcl-2和VEGF表达,可能对早产儿视网膜病变等视网膜新生血管疾病具有治疗作用。     

血管内皮生长因子反义寡聚脱氧核苷酸对增生性视网膜病变幼鼠血管内皮生长因子表达的影响

目的 观察血管内皮生长因子（vascular endothelial cell growth factor,VEGF）反义寡聚脱氧核苷酸（antisense oligodeoxynucleotides，ASODNs）对增生性视网膜病变幼鼠模型VEGF表达的影响。 方法 将30只Sprague-Dawley(SD) 新生幼鼠随机分为正常对照组、用药组及未用药组,每组10只。用高浓度氧诱导用药组及未用药组SD幼鼠，建立增生性视网膜病变幼鼠模型。对用药组幼鼠行VEGF ASODNs 球后注射，3d后取各组幼鼠除角膜、巩膜及晶状体外的全部眼内组织及血清，通过竞争性酶免疫测定法检测其VEGF的含量。 结果 用药组幼鼠眼内组织VEGF含量明显低于未用药组（P＜0.05），而与对照组相比差异无显著性的意义（P＞0.05）；用药组幼鼠血清VEGF含量与未用药组相比差异无显著性的意义（P＞0.05），两者均明显低于对照组（P＜0.05）。 结论 VEGF ASODNs能明显抑制由高浓度氧诱导的增生性视网膜病变幼鼠VEGF的表达。 （中华眼底病杂志，2003，19：172-174）     

内皮抑素对视网膜新生血管中血管内皮细胞生长因子表达的影响

目的:观察内皮抑素(endostatin,ES)对视网膜新生血管中血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法:通过缺氧的方法建立小鼠视网膜新生血管模型36只,随机分为高氧组及ES治疗组。ES治疗组小鼠在出氧箱后12,36h,玻璃体腔内注射ES1μL。另将生活在正常氧环境中的18只同龄小鼠作为正常对照。提取3组小鼠视网膜总RNA,通过RT-PCR方法定量检测VEGF在3组小鼠视网膜中的表达。结果:氧致视网膜病变视网膜中VEGF的表达升高,与正常对照组比较有统计学意义(高氧组与正常对照组比较P<0.05);ES作用后VEGF的表达减少,与给氧组比较有统计学意义(ES治疗组与高氧组比较P<0.05)。结论:ES作用机制可能是通过抑制VEGF mRNA基因的表达来抑制视网膜新生血管的形成。     

血管内皮生长因子抑制剂在糖尿病性视网膜病变中的应用

眼部新生血管是糖尿病性视网膜病变致盲的主要病理改变,而血管内皮生长因子（vascular endothelial growth factor,VEGF）在新生血管形成过程中起关键性刺激作用。VEGF抑制剂主要通过与VEGF结合并阻断其生物活性而起作用,从而达到抑制眼部新生血管生成的目的,在糖尿病性视网膜病变血管渗漏及新生血管形成的治疗中取得了显著的成绩。Bevacizumab（Avastin）是VEGF抑制剂之一,属于重组人源化单克隆抗体,因其疗效良好、价格低廉已被广泛应用于临床。现将VEGF抑制剂（尤其是Avastin）治疗糖尿病性视网膜病变的相关应用进展作一综述。     

曲安奈德玻璃体腔注射对氧诱导视网膜新生血管的抑制作用及机制

目的 观察曲安奈德(TA)玻璃体腔注射对氧诱导视网膜新生血管的抑制作用,探讨其作用机制.方法 7日龄C57BL/6新生小鼠48只,随机分为正常对照组、单纯高氧组、TA正常组及TA高氧组,分别为6、6、18、18只.单纯高氧组及TA高氧组建立氧诱导视网膜新生血管模型.选取TA正常组及TA高氧组小鼠1只眼,玻璃体腔注射20μg/μl的TA 2 μl;对侧眼注射相同体积的平衡盐溶液(BSS)作为BBS正常组和BBS高氧组.小鼠17日龄时,各组作石蜡切片苏木精-伊红(HE)染色,观察并统计每张切片突破视网膜内界膜的血管内皮细胞核数.TA正常组、BSS正常组、TA高氧组及BSS高氧组作石蜡切片免疫组织化学染色,检测血管内皮生长因子(VEGF)、基质细胞衍生因子1(SDF-1)及CD14的平均吸光度[A,旧称光密度(OD)]值.并行荧光实时定量聚合酶链反应(PCR),检测VEGF及SDF-1的mRNA表达.结果 正常对照组、单纯高氧组、TA正常组、BSS对照组、TA高氧组及BSS高氧组突破视网膜内界膜的血管内皮细胞核数分别为0、675、0、0、110及688个.正常对照组较单纯高氧组明显减少,差异有统计学意义(t=30.62,P＜0.05).TA高氧组较BSS高氧组显著减少,差异有统计学意义(t=19.532,P＜0.05).TA正常组与BSS正常组VEGF、SDF-1及CD14平均A值比较,差异无统计学意义(t=0.161,0.284,0.223;P＞0.05).TA高氧组VEGF、SDF-1及CD14平均A值较BSS高氧组减少,差异有统计学意义(t=-2.264,-2.358,-4.897;P＜0.05).TA正常组与BSS正常组VEGF及SDF-1 mRNA表达比较,差异无统计学意义(t=-0.497,-0.709;P＜0.05).TA高氧组与BSS高氧组VEGF及SDF-1 mRNA表达比较,差异均有统计学意义(Z=-5.137,-4.411;P＜0.05).结论 玻璃体腔注射TA能有效抑制氧诱导视网膜新生血管形成,其抑制作用可能是通过降低VEGF及SDF-1的表达而实现.     

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.     

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。     

Bevacizumab treatment reduces retinal neovascularization in a mouse model of retinopathy of prematurity

AIM: To evaluate the effect of different bevacizumab concentrations on retinal neovascularization in a retinopathy of prematurity (ROP) mouse model.METHODS: A total of 60 of C57BL?6 J mice were exposed to 75%±2% oxygen from postnatal d7 to postnatal d12. Fifteen nonexposed mice served as negative controls (group A). On d12, 30 mice (group C) were injected with 2.5 µg intravitreal bevacizumab (IVB), 30 mice (group D) were injected with 1.25 µg IVB in one eye. The contralateral eyes were injected with balanced salt solution (BSS) (control group=group B). The adenosine diphosphatase (ADPase) histochemical technique was used for retinal flat mount to assess the oxygen-induced changes of retinal vessels. Neovascularization was quantified by counting the endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina. Histological changes were examined by light microscopy. The mRNA levels of vascular endothelial growth factor (VEGF) were quantified by Real-time PCR. Western-blotting analysis was performed to examine the expression of P-VEGFR.RESULTS:Comparing with the control group B, regular distributions and reduced tortuosity of vessels were observed in our retinal flat mounts in groups C and D. The endothelial cell count per histological section was lower in groups C (P<0.0001) and D (P<0.0001) compared with the control group B. Histological evaluation showed no retinal toxicity in any group. In all oxygen treated groups VEGF mRNA expression was significantly increased as compared to age-matched controls. No significant change in VEGF mRNA expression could be achieved in either of the treatments or the oxygen controls. The results of the Western blot were consistent with that of the Real-time PCR analysis.CONCLUSION:An intravitreal injection of Bevacizumab is able to reduce angioproliferative retinopathy in a mouse model for oxygen-induced retinopathy.     

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。     

Inhibition of LY294002 in retinal neovascularization via down-regulation the PI3K/AKT-VEGF pathway in vivo and in vitro

AIM: To investigate the effects of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 on retinal neovascularization (RNV) in the oxygen-induced retinopathy (OIR) mouse model and human umbilical vein endothelial cells (HUVECs). METHODS: C57BL/6J mice were randomly divided into normoxia-control, OIR-control and LY294002 treatment groups. LY294002 or phosphate-buffered solution was intraperitoneally injected daily into mouse pups from P6 to P9 in LY294002 treatment group or OIR-control group. Morphological and pathological changes in RNV, as well as expression levels of PI3K, serine-threonine kinase (AKT) and vascular endothelial growth factor (VEGF) were observed. HUVECs treating with LY294002 were exposed to hypoxia; the expression of PI3K, AKT and VEGF were examined by Western blot and RT-PCR analyses. RESULTS: Compared with the OIR-control group, LY294002 significantly inhibit RNV. Adenosine diphosphatase (ADPase) staining and hematoxylin and eosin staining indicated that the clock hour scores of neovascularization and the nuclei of pre-retinal neovascular cells in the LY294002 treatment group were clearly less than those in the OIR-control group (1.41±0.52 vs 6.20±1.21; 10.50±1.58 vs 22.25±1.82, both P<0.05). Intravitreal injection of LY294002 (in the LY294002 treatment group) markedly decreased PI3K/AKT-VEGF expression compared with the OIR-control group by immunohistochemistry, Western blotting and RT-PCR (all P<0.05). In HUVECs treated with hypoxia, expression of PI3K, AKT and VEGF were downregulated in the hypoxia-LY294002 group (all P<0.05). CONCLUSION: The PI3K inhibitor LY294002 can inhibit RNV by downregulating PI3K, AKT, and VEGF expression in vivo and in vitro. LY294002 may provide an effective method for preventing retinopathy of prematurity (ROP).