内皮抑素抑制脉络膜新生血管及VEGF和bFGF表达的研究

目的 研究内皮抑素对实验性脉络膜新生血管的抑制作用。方法 50只雄性BN大鼠，40只为实验组，10只为空白对照组。用半导体激光光凝实验组BN大鼠的视网膜，随机分为4组，内皮抑素Ⅱ组、内皮抑素Ⅰ组、生理盐水组和激光损伤组，每组10只；由光凝后当天开始，至光凝后13d，每天分别给予腹腔注射内皮抑素20mg／kg、10mg／kg和生理盐水1.2mL，激光损伤组光凝后不做任何处理。空白对照组为未行激光光凝和给药处理的BN大鼠。于光凝后第7天及第14天行眼底血管荧光素造影检查、组织病理学检查、VEGF免疫组化检测和bFGF mRNA的原住杂交检测。结果随着内皮抑素剂量增加，荧光素渗漏率和渗漏强度减弱；激光光凝后7d，内皮抑素Ⅱ组与其他组相比，渗漏强度的差异有显著性；光凝后14d，两个内皮抑素组与生理盐水组和激光损伤组比较，荧光素渗漏强度差异均有显著性；生理盐水组和激光损伤组差异无显著性。激光光凝后7dVEGF和bFGFmRNA表达达高峰，光凝后14d下降，内皮抑素组vEGF和bFGFmRNA表达较激光损伤组减弱，并呈剂量依赖性趋势。结论 内皮抑素可以有效地抑制实验性CNV的形成。下调VEGF和bFGF的表扶可能是肉皮抑素抑制CNV的作用机制之一．     

实验性兔脉络膜新生血管模型研究

目的:探讨氩激光创建有色家兔脉络膜新生血管(choroidal neovascularization,CNV)模型的可行性,初步探讨CNV的有关发生机制,为其进一步防治研究奠定基础.方法:健康有色家免20只(40只限),每只兔一眼为实验眼,另眼为对照眼.以波长514.5 nm的氩绿激光(光斑直径50um,功率0.7W,时间0.1s),在距视盘2～3个视盘直径的颢侧髓线上下视网膜处密集照射20个点.分别于光凝后3,7,14,21,30天进行眼底荧光造影(fundus fluorescein angiography,FFA)及脉络膜造影(indocyanine green angiography,ICGA).检查后处死动物(4只/次),摘除眼球取眼后段组织制作石蜡切片.常规HE染色及用免疫组化(S-P法)检测血管内皮生长因子(VEGF)在视网膜的表达.结果:光凝后7天出现CNV,兔CNV发生率尚(63.8%),眼底造影和光镜下均有相应改变,FFA表现为视网膜血管无关的荧光素渗漏,ICGA见CNV充盈;光镜下可见到CNV结构;光凝后2周开始VEGF在视网膜神经节细胞以及内核层有表达.结论:氩激光诱导有色家免CNV模型成模时间短,成模率较高;VEGF参与了CNV的形成进程.     

视瞻昏渺发病机理与VEGF、bFGF、PEDF表达

目的：观察脉络膜新生血管（CNV）动物模型视网膜色素上皮至脉络膜之间细胞因子VEGF、bFGF、PEDF表达的变化。方法：用半导体激光光凝建立CNV动物模型，于造模后1W，2W，3W，4W，8W分别行免疫荧光检查（FFA）；于造模后8W处死动物行HE染色观察视网膜和脉络膜的病理组织学变化，免疫组化法检测VEGF、bFGF、PEDF表达。结果：HE染色可见脉络膜新生血管形成。造模后模型组VEGF、bFGF上调，VEGF／PEDF值升高，与正常对照组比较，差异有统计学意义（P〈0．01）；PEDF值轻度上调，与正常对照组比较差异无统计学意义（P〉0．05）。结论：半导体激光可诱导CNV动物模型，该模型可做为视瞻昏渺的动物模型进行实验研究。     

半导体激光诱导大鼠脉络膜新生血管模型的建立

目的 探讨应用半导体激光诱导建立BN大鼠脉络膜新生血管(choroidal neovascularization，CNV)模型的可行性。方法 雄性棕色挪威(Brown Norway，BN)大鼠30只，9只为对照组，21只为实验组；用半导体激光光凝实验组BN大鼠的视网膜，分别于光凝后1h，3、7及14d行眼底荧光造影(fiundus fluorescein angiography，FFA)检查，3、7和14d行病理组织学检查。结果 光凝后7dFFA及光镜检查均证实CNV开始形成。14dCNV大量形成。结论 半导体激光损伤可以成功地诱导BN大鼠形成CNV模型，成模时间短，成模率高，是一种较为理想的CNV动物模型。     

Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization

Background Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization （CNV）, and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor （PEDF） could inhibit CNV.
Methods Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDFgene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure （intravitreal injection）; infusing liposomes with the naked plasmid DNA of PEDF into the vitreous （lipofectamine ＋ PEDF）; infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately （retrobular injection）; infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately （vein injection）. The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography.
Results In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively.
Conclusions Ultrasound-microbubble technique could increase PEDF gene transfer into rats＇ retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.     

核蛋白NSA2在脉络膜新生血管动物模型眼组织的表达研究

目的:NSA2是1种新发现的与细胞增殖相关的核蛋白,本实验旨在初步探讨NSA2是否参与了脉络膜新生血管(Chor-oidal neovascularization,CNV)病变过程.方法:采用532 nm波长的眼底激光对16只小鼠光凝处理,诱导CNV动物模型;小鼠眼底激光光凝处理5d后,去除眼前段,定量PCR检测NSA...     

抑制kB活性对碱烧伤大鼠角膜新生血管内皮生长因子表达的影响

目的观察碱烧伤后大鼠角膜组织核转录因子-kB(NF-kB)和血管内皮生长因子(VEGF)的变化,以及抑制NF-kB活性对碱烧伤大鼠角膜组织新生血管VEGF表达的影响。方法建立大鼠角膜碱烧伤诱发CNV模型,采用裂隙灯、免疫组化等方法,分别检测使用NF-kB活性抑制剂前后VEGF在SD大鼠CNV中的表达情况。结果在正常角膜,VEGF没有表达或在上皮基底膜有微弱表达;在新生血管对照组,角膜全层VEGF表达明显增强;用药组VEGF表达减少。经SPSS软件分析,三组间具有统计学差异。结论抑制NF-kB的表达可降低VEGF的表达。     

Rescue of retinal function by macular translocation surgery in age-related macular degeneration and other diseases with subfoveal choroidal neovascularization

Age-related macular degeneration (AMD) is the leading cause of legal blindness among the elderly. In AMD and some other macular diseases, subfoveal choroidal neovascularization (CNV) damages the underlying retinal pigment epithelium (RPE), and because retinal function is dependent on a healthy RPE, vision is markedly reduced by a subfoveal CNV. To treat such CNVs, macular translocation surgery has been performed to move the sensory retina from the damaged RPE to healthier RPE. At present, this surgery is the only possible treatment to improve the visual acuity of patients with subfoveal CNV. Macular translocation surgery involves the detachment of the entire retina from the RPE by a subretinal infusion of fluid and creating a 360 degrees circumferential retinotomy followed by the rotation of the retina. Severe postoperative complications such as recurrent retinal detachment have been reported in about 30% of the cases after macular translocation. To determine the efficacy of this surgery, it is necessary to demonstrate an improvement in macular and overall retinal function objectively as well as subjectively. To this end, we have assessed the changes in visual function by measuring the visual acuity subjectively, and the macular function objectively by focal macular ERGs (FERGs). We shall show that there is an improvement in the FERGs in most patients after retinal translocation surgery but the full-field ERGs were reduced by about 30%. Thus, macular translocation surgery with 360-degree retinotomy may be feasible for macular function, although some degree of peripheral retinal function is lost.     

环氧化酶-2和VEGF在角膜新生血管中的表达和相关性研究

目的观察环氧化酶-2（COX～2）和血管内皮生长因子（VEGF）在碱烧伤诱导的兔角膜新生血管（CNV）中的表达，分析其相关性，探讨CNN形成的有关机制。方法采用碱烧伤建立兔CNV模型，伤后每日裂隙灯显微镜观察CNV生长情况并计算其面积，并于第3、7、10、14天各时段，免疫组化S—P法检测COX-2和VEGF蛋自在角膜组织中的表达，分析其相关性。结果CNV生长面积在伤后第3、7、10、14天逐渐增大，COX-2和VEGF的表达逐渐增加，主要分布于炎症细胞胞质及新生血管内皮细胞，相关性分析显示二者呈显著正相关（r=0．995，P〈0．05）。结论COX-2和VEGF参与炎症相关CNV形成，其机制可能与COX-2上调VEGF表达有关。     

整合素αvβ3、组织因子及血管内皮细胞生长因子在实验性脉络膜新生血管中的表达

目的：探讨整合素αvβ3、组织因子(tissue factor, TF)及血管内皮细胞生长因子（vascular endothelial growth factor, VEGF）在激光诱导的大鼠脉络膜新生血管（choroidal neovascularization, CNV）组织中的表达。方法：随机选取成年雄性棕色挪威大鼠25只,一眼为实验组,经氩激光（波长532 nm）光凝诱导CNV形成,另一眼为正常对照。分别于光凝后第3,7,14,21及28天随机取5只大鼠,行眼底照相、眼底荧光血管造影（fluorescence fundus angiography, FFA）检查,并取出视网膜,通过免疫组织化学方法检测整合素αvβ3,TF及VEGF在CNV中的表达及相互关系。结果：正常对照组无新生血管生成,无整合素αvβ3和TF的表达,但视网膜神经节细胞层、内核层、色素上皮层、视网膜及脉络膜血管内皮细胞中均有少量VEGF的表达。光凝7 d后, FFA检查发现实验眼光凝区有圆盘状荧光渗漏,表明有新生血管生成。光凝后3 d,光凝区视网膜神经节细胞层、内核层、外核层缺损区、视网膜及脉络膜血管内皮细胞均表达少量的VEGF、整合素αvβ3及TF,随着时间的延长,CNV逐渐形成,VEGF、整合素αvβ3及TF表达水平也逐渐增加(P<0.01),其中整合素αvβ3的表达于第7天达高峰;VEGF的表达于第14天达高峰;TF的表达于第21天达高峰。结论：整合素αvβ3,VEGF及TF分别表达于实验性大鼠CNV形成初期、中期及晚期,其在CNV中表达的顺序对临床选择抗新生血管药物的治疗时间具有十分重要的意义。     

奥曲肽联合经瞳孔温热疗法治疗实验性脉络膜新生血管

目的观察奥曲肽或联合经瞳孔温热疗法(TTT)对大鼠脉络膜新生血管(CNV)的疗效。方法使用激光诱导大鼠CNV,光凝术后7d球后注射磷酸缓冲盐溶液(PBS组,n=6）、奥曲肽50μg/kg（n=6）以及奥曲肽50μg/kg联合TTT（n=6）,另取3只大鼠未给与激光及球后注射,用于实时定量PCR（qRT PCR）检测。光凝术后14?d采用眼底荧光血管造影观察CNV的荧光渗漏变化,脉络膜铺片定量分析CNV面积变化,qRT PCR观察RPE 脉络膜复合物血管内皮生长因子(VEGF)和胰岛素样生长因子-1(IGF-1) mRNA表达水平的变化。结果奥曲肽或奥曲肽联合TTT能有效抑制CNV荧光渗漏,使CNV面积减小,VEGF和IGF-1 mRNA水平较PBS组明显降低(P<0.05),联合疗法的抑制作用更为显著(P<0.05)。结论奥曲肽联合TTT为治疗CNV提供了一条新的途径。     

经瞳孔温热疗法治疗年龄相关性黄斑变性的脉络膜新生血管的疗效观察

目的观察经瞳孔温热疗法（transpupillary thermotherapy，TTT）治疗年龄相关性黄斑变性（age—related macular degeneration，ARMD）的脉络膜新生血管（choroidal neovascularization，CNV）的疗效。方法12例12眼ARMD患者行TTT治疗，根据CNV的大小选择不同的光斑大小和能量，照射时间为1min。结果12眼ARMD经TTT治疗后，视力无明显提高，但黄斑出血、渗出、水肿减轻或吸收，眼底血管荧光素造形（FFA）显示大多数CNV渗漏减少或停止，原CNV瘢痕化。结论TTT治疗ARMD的脉络膜新生血管有效，但还需要更多的病例来评价治疗效果。     

杞黄颗粒对实验性脉络膜新生血管VEGF蛋白及其mRNA表达的影响

[目的]探讨杞黄颗粒对激光诱导脉络膜新生血管(CNV)大鼠血管内皮生长因子(VEGF)蛋白及其mRNA表达的影响。[方法]81只雄性棕色挪威(BN)大鼠,采用随机分组法,13只为空白对照组;68只造模,随机抽取3只验证CNV形成,剩余65只随机分为5组:杞黄颗粒A组(简称杞黄A组),杞黄颗粒B组(简称杞黄B组),雷珠单抗组,杞黄颗粒+雷珠单抗组(简称杞黄+雷珠组),模型组。除杞黄B组给药90 d,其余组给药60 d后检测VEGF蛋白含量和mRNA表达水平。[结果]造模第7天,CNV验证形成。用药60 d,杞黄+雷珠组与杞黄A组、模型组相比VEGF的蛋白含量也明显降低,均P0.05,具有统计学差异。杞黄B组与杞黄A组相比,VEGF蛋白含量降低降低,P0.05,具有统计学意义。用药60 d,空白对照组相比模型组,杞黄+雷珠组相比模型组VEGF mRNA均有明显降低,P0.05,具有统计学差异;杞黄B组相比杞黄A组的VEGF mRNA降低明显,P0.05,具有统计学意义。[结论]杞黄颗粒与雷珠单抗联合使用,对于VEGF的表达有明显的调节作用,能够减少CNV的形成,对湿性老年性黄斑变性(WAMD)有一定治疗意义。     

脉络膜新生血管患者房水中血管内皮生长因子和色素上皮衍生因子的浓度

目的:研究血管内皮生长因子(VEGF)和色素上皮衍生因子(PEDF)在活动性脉络膜新生血管膜(CNV)患者房水中的表达。方法:应用酶联免疫吸附测定法对32例活动性CNV患者和10例白内障患者(对照组)的房水标本进行VEGF和PEDF检测。结果:CNV患者房水中的VEGF为(523.0±273.7)pg/m l,PEDF为(16.58.±13.11)ng/m l;对照组患者房水中的VEGF为(108.3±72.3)pg/m l,PEDF为(0.35±0.57)ng/m l。两组间的VEGF和PEDF均有显著性差异(均P=0.000)。结论:活动性CNV患者房水中VEGF和PEDF增高,可能与脉络膜新生血管的形成有关。     

血管内皮生长因子在氩激光诱导大鼠脉络膜新生血管中的表达

目的:探讨血管内皮生长因子(VEGF)在氩激光诱导BN大鼠脉络膜新生血管(CNV)形成过程中的表达及其意义。方法:BN大鼠18只,随机分成3组,每组6只,单眼视网膜氩激光光凝,诱导CNV形成。分别在光凝后1周、2周、3周摘除眼球,应用免疫组织化学技术对光凝区进行因子Ⅷ相关抗原(FⅧR:Ag)的检测以观测CNV形成,并检测CNV形成过程中VEGF蛋白的表达,分析其变化趋势。结果:FⅧR:Ag免疫组织化学检查结果显示,光凝后1周开始形成CNV,3周达到高峰。正常BN大鼠视网膜中,VEGF在视网膜神经节细胞层、内核层、色素上皮层、视网膜和脉络膜血管内皮细胞表达。视网膜光凝后,除上述部位外,VEGF在外核层和脉络膜的光凝损伤区阳性表达。光凝后1周~3周,光斑区内FⅧR:Ag、VEGF阳性染色密度逐渐增加(P<0.05),FⅧR:Ag与VEGF阳性染色密度正相关(r=0.83,P<0.05)。结论:CNV形成与VEGF表达正相关,VEGF表达增多可能是CNV形成和增殖的主要原因之一。     

COX-2在角膜新生血管中的表达及血管形成抑制实验研究

目的:检测COX-2、VEGF在角膜新生血管形成中的表达及探讨COX-2选择性抑制剂对新生血管的抑制作用.方法:建立碱烧伤致大鼠角膜新生血管模型,通过裂隙灯和病理学观察,免疫组化方法检测COX-2和VEGF的表达.结果:阳性组见大量炎性细胞浸润,角膜新生血管丰富,用药组的上述改变明显减轻,且术后各时间点COX-2和VEGF表达均较阳性组明显减弱,有显著性差异(P<0.01).二者表达变化与病理组织学表现相平行.且COX-2与VEGF呈显著正相关(P<0.05).结论:COX-2和VEGF参与了角膜新生血管的形成过程,COX-2选择性抑制剂可抑制和延缓新生血管的发生、发展.     

脉络膜新生血管单次光动力治疗后黄斑区视功能与形态变化

目的观察单次光动力治疗(PDT)脉络膜新生血管(CNV)后黄斑区视功能与形态变化及其相关性。方法选择17例(23眼)经典为主型CNV患者进行单次光动力治疗,于治疗前及治疗后3个月和8个月分别行视力、多焦视网膜电图(mfERG)、荧光素眼底血管造影(FFA)、相干光视网膜断层扫描(OCT)检查,观察黄斑中心的厚度以及功能改变;选择相匹配的正常30眼作为对照组。结果与对照相比,CNV患眼治疗前黄斑中心厚度增加,且mfERG的N1和P1波反应密度明显下降。PDT治疗后,视力有改善趋势,但差异无统计学意义。OCT显示治疗后黄斑区中心厚度显著减小,但治疗后8个月与治疗后3个月差异无统计学意义。mfERG显示治疗后1区P1波和2区N1波反应密度显著增加;治疗后8个月1区N1波反应密度显著增加;1区和2区的P1和N1潜伏期治疗前后差异无统计学意义。治疗后3个月视力变化与黄斑中心厚度变化有相关性(r=0.43,P=0.04),与mfERG各波的变化无相关性,1区的N1和P1波反应密度与黄斑中心厚度变化呈负相关(r=-0.43,P=0.04;r=-0.51,P=0.01)。结论对较小范围的CNV患眼单次PDT治疗可在3个月内减少黄斑区中心厚度并改善视功能,并在相对远期保持稳定。黄斑中心区和旁中心区的恢复有所不同;视功能变化与形态变化有一定相关性。     

光动力疗法治疗病理性近视脉络膜新生血管的临床观察

目的 探讨病理性近视脉络膜新生血管采用光动力疗法（PDT）治疗的临床效果.方法 选择病理性近视脉络膜新生血管患者20例（20眼）,均采用PDT治疗,回顾性分析治疗前后临床资料.结果 末次随访时,检测视力呈＞2行提高者3眼,占15％;保持稳定者16眼,占80％;视力下降1眼,占5％.眼底检查示视网膜神经感觉层缩小或脱离,黄斑渗出、水肿、出血均有程度不等的吸收.视网膜出血在治疗6个月后基本吸收.眼底荧光血管造影（FFA）/吲哚青绿血管造影（ICGA）在治疗后3个月检查显示,12只眼黄斑下脉络膜新生血管（CNV）荧光素渗漏静止,CNV闭合占60％,9只眼CNV渗漏好转,占45％.结论 CNV采用PDT治疗可获得理想效果,因为短期观察,选取的病例数相对较少,而从根本上,PDT无法杜绝CNV发生,故存在未彻底治疗的情况,长时间不能完全消失,也不能改善远期视力,若需客观评价PDT疗法,需行远期观察.     

Blockade of the sonic hedgehog signalling pathway inhibits choroidal neovascularization in a laser-induced rat model

Sonic hedgehog (Shh) signaling has recently been shown to be involved in the pathological angiogenesis in response to tissue hypoxia and ischemic injury.Hypoxia/ischemia is considered to play an important role in the development of choroidal neovascularization (CNV).This study was aimed to examine the effect of blockade of the Shh signaling pathway on CNV and the underlying mechanism.A total of 64 male Brown-Norway (BN) rats were used in this study.One eye of each rat underwent laser photocoagulation.The other eye served as normal control.After the laser treatment, the 64 rats were divided into four groups (n=16 in each group):Blank control group, in which no intravitreal administration was given; cyclopamine group, recombinant Shh N-terminals protein (rShh) group and phosphate-buffered saline (PBS) group, in which cyclopamine (a Shh inhibitor), rShh (a Shh activator) and PBS were intravitreally injected into the laser-treated eyes respectively every other day for a total of four intravitreal injections immediately after the laser treatment.Fourteen days after the intravitreal administration, the changes of CNV-related variables, including positive CNV lesion percentage, CNV membrane area and CNV membrane thickness, were evaluated by fluorescein anqiography, indocyanine green angiography and pathological examinations.The mRNA and protein expression of PTCH1, Gli1, HIF-1α, VEGF and DLL4 in each group on 14 days of CNV model was detected by real-time quantitative PCR and western blot analysis, and the relationship between the Shh cascade and the HIF-1α-VEGF-DLL4 cascade in CNV was analyzed.The results showed that the CNV membrane area and the CNV membrane thickness were decreased by 62.5% and 41.9% in the cyclopamine group and increased by 85.7% and 64.3% in the rShh group in comparison to those in the blank control group (P<0.01 for each).There was no significant difference in the CNV membrane area and thickness between the blank control group and PBS group (P=0.102 and P=0.063, respectively).Real-time quant     

MMP-2、TIMP-2与碱烧伤后角膜新生血管形成的实验研究

目的:探讨基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-2组织型抑制剂(TIMP-2)在碱烧伤大鼠角膜新生血管(CNV)形成中的表达和意义。方法:采用碱烧伤大鼠角膜建立CNV模型,摘除角膜作病理切片,多形核白细胞(PMN)记数;免疫组化法检测MMP-2、TIMP-2的表达。结果:烧伤后1d角膜缘PMN开始增多,到CNV7d组PMN增加最明显,此后逐渐减少;免疫组化显示:MMP-2在CNV中阳性表达逐渐增加,于7d表达最明显,此后随炎性细胞的减少而减弱,21d后几乎无表达;TIMP-2则于早期变化不明显,7d表达开始升高,14d达高峰。结论:烧伤后CNV形成早期,MMP-2活性增高,继而TIMP-2表达增加,使MMP-2活性受抑,基底膜降解受阻,新生血管延伸停滞;CNV形成中,MMP-2的表达增加,并与角膜的炎性反应程度一致,PMN浸润可能是CNV形成的关键因素。