血管内皮生长因子反义核苷酸抑制大鼠角膜新生血管的实验研究

目的探讨重组腺相关病毒介导的血管内皮生长因子反义核苷酸(rAAV aVEGF165)抑制大鼠角膜新生血管(CNV)发生与发展的作用。方法三质粒共转染法构建rAAV aVEGF165和重组腺相关病毒介导的β半乳糖苷酶的结构基因(rAAV Lacz)。48只SD大鼠用抽签法随机分成实验组和对照组,每组24只,碱烧伤方法诱导CNV生成。实验组结膜囊注射rAAV aVEGF165(1010pfu/ml),对照组结膜囊注射rAAV Lacz(1010pfu/ml);5溴4氯3吲哚βD半乳糖苷(X gal)组织化学染色,检测Lacz基因在结膜囊中的表达情况;碱烧伤后1、2、4、7、14及30d,形态学分析评价角膜新生血管的生长情况,免疫组织化学检测角膜组织中VEGF的表达。结果对照组rAAV Lacz结膜囊注射2d后,检测Lacz基因在结膜组织中的表达效率为21.36%±1.07%;30d后Lacz基因在结膜下组织中的表达效率为28.02%±1.16%。碱烧伤后结膜囊注射rAAV aVEGF165,实验组CNV面积明显减少,新生血管密度降低,VEGF在角膜组织中的表达明显降低,差异有统计学意义(F=1639.22,F=2187.16,F=719.17,P<0.01)。结论rAAV aVEGF165能显著抑制碱烧伤后CNV的形成,其机制与降低角膜组织中VEGF的表达有关,重组腺相关病毒是眼反义基因治疗的有效载体。     

角膜碱烧伤后VEGF的表达与新生血管的关系

目的研究碱烧伤后角膜血管内皮生长因子（vascular endothelial growthfactor，VEGF）的表达与角膜新生血管化的关系。方法30只Sprague-Dawleg（SD）大鼠碱烧伤，诱导角膜新生血管模型。碱烧伤后不同时间形态学分析来评价角膜新生血管的情况，免疫组化及Westem blot评价角膜VEGF的表达。结果角膜碱烧伤后24hVEGF的表达开始增高，伤后4d达高峰，伤后7d VEGF的表达下降，14d后显著下降。碱烧伤后，角膜新生血管与VEGF的表达呈明显的平行关系。结论碱烧伤后大鼠角膜VEGF的表达水平与新生血管的形成有相关性，并在其中发挥重要作用。     

血管内皮生长因子shRNA表达质粒抑制鼠角膜新生血管与血管内皮细胞中血管内皮生长因子的表达

目的 探讨RNA干扰对大鼠碱烧伤后角膜新生血管(CNV)与血管内皮细胞(EC)中血管内皮生长因子(VEGF)表达的影响及作用机制.方法 实验研究.采用DNA重组技术设计合成针对VEGF mRNA的3条shRNA寡核苷酸片段,构建真核表达质粒VEGF shRNA1、2、3,然后将质粒通过脂质体介导分别转染EC,逆转录聚合酶链反应和Western blot免疫印迹法分别检测EC中VEGF mRNA和蛋白的表达及干扰效率,并筛选出干扰效率最高的质粒(VEGF shRNA3质粒)进行体内实验.将20只SD大鼠按完全随机设计方法分为两组,分别为实验组10只和对照组10只,建立大鼠碱烧伤后CNV模型.实验组于结膜下注射VEGF shRNA3质粒后,显微镜下观察CNV生长情况,7 d后利用逆转录聚合酶链反应(RT-PCR)与Western blot法分别检测实验组和对照组角膜组织VEGF mRNA和蛋白的表达.多组间差异显著性检验用单因素方差分析,两组CNV面积比较采用重复测量的方差分析.结果 经酶切及测序证实重组质粒构建成功.VEGF shRNA1、2、3质粒均可下调VEGF mRNA和蛋白的表达,其中VEGF shRNA3质粒的抑制作用较强,VEGF mRNA和蛋白的抑制率分别为68.92%和66.22%.体内实验中碱烧伤后3、5、7 d,实验组CNV长度较对照组明显降低,差异有统计学意义(F=355.744,P=0.000);实验组CNV面积较对照组明显降低,差异有统计学意义(F=402.700,P=0.000);实验组VEGF mRNA和蛋白在角膜组织中的表达较对照组明显降低,抑制率分别为41.84%和41.86%.结论 构建的VEGF shRNA质粒能右效抑制EC中VEGF mRNA和蛋白的表达.结膜下注射VEGF shRNA3质粒能显著抑制碱烧伤后CNV的形成与VEGF的表达.     

Bevacizumab对大鼠角膜新生血管的抑制作用

目的：评价Bevacizumab抑制大鼠角膜新生血管的效果。 方法：碱烧伤诱导角膜新生血管模型，对20只wistar大鼠40眼随机平均分4组。角膜碱烧伤后分别给予结膜下注射药物溶剂和不同剂量Bevacizumab。比较并计算角膜新生血管生长面积。16d后角膜标本行HE检验和免疫组织化学检测VEGF的表达。 结果：各用药组与对照组新生血管面积比较差异有统计学意义；治疗组间新生血管面积比较差异没有统计学意义。组织学发现各药物治疗组炎性细胞浸润、新生血管形成均明显轻于对照组。新生血管对照组VEGF染色明显增强，药物注射组表达明显减弱。 结论：结膜下注射一定浓度的Bevacizumab对大鼠角膜碱烧伤形成的新生血管有抑制作用。     

Bevacizumab对大鼠角膜新生血管的抑制作用

目的 观察bevacizumab对角膜新生血管的抑制作用.方法 选取鼠龄6～8周、体质量(180±10)g的雄性Wistar大鼠40只,建立碱烧伤角膜新生血管模型;将大鼠随杌分为3个不同剂量药物治疗组(实验组)和1个对照组,每组10只(20眼),大鼠角膜碱烧伤后分别给予结膜下注射不同剂量(0.5 mg、1.0 mg、2.0 mg)的bevacizumab,对照组注入生理盐水;然后进行角膜新生血管的各项数据观察,观察期为16 d,主要观察项目包括各组角膜新生血管的生长情况;计算角膜新生血管的生长面积;碱烧伤后第7天和第16天后采集角膜,标本行组织病理学检测和免疫组织化学检测,第16天,计算平均OD值;评价药物对角膜新生血管的抑制效果.结果 碱烧伤后第7天、第14天,实验组与对照组新生血管面积比较差异均有统计学意义(均为P＜0.05);组织病理学检测发现各实验组炎性细胞浸润、新生血管形成均明显轻于对照组.对照组血管内皮生长因子(vascular endothelial growth factor,VEGF)染色明显增强,实验组的表达明显减弱.碱烧伤后第16天,实验组与对照组比较,VEGF染色阳性细胞数和VEGF平均OD值差异均有统计学意义(均为P＜0.05).碱烧伤后第7天、第16天大鼠角膜CD34的免疫组织化学检测结果及新生血管密度计数方面,实验组和对照组比较差异均有统计学意义(均为P＜0.05).结论 结膜下注射一定浓度的bevacizumab对大鼠角膜碱烧伤后的新生血管生长有抑制作用.     

VEGF siRNA对兔眼碱烧伤后角膜新生血管的抑制作用

目的:研究血管内皮生长因子小干扰RNA(VEGF small in-terfering RNA,VEGF siRNA)对兔眼碱烧伤后角膜新生血管的抑制作用。方法:普通家兔25只以碱烧伤法(1mol/LNaOH溶液)诱导角膜新生血管(CNV)生成。碱烧伤后立即以脂质体(LF2000)为载体,右眼球结膜下注射VEGF siRNA重组质粒,左眼球结膜下注射pSilencer 2.1-U6 hygro空白质粒作为阴性对照。碱烧伤后1,3,5,7,14d,形态学分析评价角膜新生血管的生长情况。结果:与对照眼相比,碱烧伤后球结膜下注射VEGF siRNA重组质粒,实验眼在各时间段(3,5,7,14d),CNV长度明显变短,面积明显变小,差异有统计学意义(P<0.05)。结论:VEGF siRNA能有效地抑制碱烧伤后CNV的形成。     

青蒿琥酯对大鼠角膜新生血管的影响

目的研究青蒿琥酯（artesunate）对大鼠角膜新生血管形成的作用。方法采用碱烧伤诱导大鼠角膜新生血管模型，将65只SD大鼠随机分成正常对照组5只（5眼）、角膜新生血管对照组30只（30眼）、青蒿琥酯球结膜下注射组30只（30眼），比较第14d角膜新生血管生长的长度和面积，并分别在烧伤后第1、3、5、7、10、14d用免疫组织化学染色的方法检测血管内皮生长因子（VEGF）、核因子κB（NF—κB）的表达。结果青蒿琥酯球结膜下注射显著抑制了角膜新生血管的生长，免疫组织化学染色VEGF、NF-κB在正常角膜上皮基底部有弱表达，在新生血管对照组有明显增强，青蒿琥酯球结膜下注射组表达明显减弱。结论青蒿琥酯球结膜下注射可以有效抑制角膜新生血管的生长，其治疗机制可能是抑制VEGF、NF—κB的表达。     

抗血管内皮生长因子小干扰RNA抑制大鼠角膜新生血管形成的研究

目的 探讨抗血管内皮生长因子小干扰RNA(VEGF-siRNA)对角膜碱烧伤急性期新生血管(CNV)形成的抑制作用.方法 实验研究.体外化学合成VEGF序列特异性双链小干扰RNA(VEGF-siRNA),脂质体介导转染体外培养的大鼠角膜上皮和基质细胞,实时PCR法检测转染后各时段VEGF mRNA表达水平,酶联免疫吸附实验测定转染后各时段VEGF蛋白表达水平.制作大鼠角膜碱烧伤动物模型,采用VEGF-siRNA转染的角膜上皮移植联合前房注射脂质体包裹的VEGF-siRNA治疗急性期CNV,免疫组化和ELISA分别检测角膜VEGF表达,观察术后CNV形成.细胞实验和动物实验中siRNA处理组和对照组VEGF蛋白含量比较,采用两样本均数比较的t检验.结果 siRNA对角膜上皮细胞VEGFmRNA的抑制效率达60%～84%,角膜基质细胞VEGFmRNA的抑制效率达59%～76%,角膜上皮和基质细胞VEGF蛋白表达也有相应的下降.采用VEGF-siRNA转染的角膜上皮移植联合前房注射VEGF-siRNA治疗,实验组CNV面积显著小于对照组;实验组VEGF表达水平显著低于对照组.结论 VEGF-siRNA能显著抑制大鼠角膜碱烧伤急性期CNV形成.     

吡格列酮对鼠角膜碱烧伤后新生血管抑制作用的研究

目的:探讨吡格列酮对大鼠碱烧伤后角膜血管内皮细胞生长因子(VEGF),碱性成纤维细胞生长因子(b-FGF)表达的调节作用,以及对碱烧伤后角膜新生血管生长的抑制作用。方法:建立大鼠角膜碱烧伤模型,结膜下注射吡格列酮,观察烧伤后不同时期的角膜新生血管的数量、长度,应用免疫组化和Western blot方法检测碱烧伤后及烧伤治疗不同时期的VEGF,b-FGF的表达变化情况,并与对照组比较。结果:大鼠角膜碱烧伤后2d角膜新生血管开始生长,7～10d达到高峰,吡格列酮治疗后的角膜新生血管的发生延迟,血管的生长受到抑制,VEGF,b-FGF随时间发生变化,表达较对照组下降。结论:局部应用吡格列酮对由碱烧伤引起的鼠角膜新生血管的发生、发展有明显的抑制作用,其机理可能是通过下调促血管生成因子VEGF,b-FGF的表达而实现的。     

兔角膜碱烧伤后VEGF的表达与新生血管的关系

目的:研究碱烧伤后角膜血管内皮生长因子(vascular endo-thelial growth factor,VEGF)的表达与角膜新生血管的关系。方法:普通家兔25只碱烧伤诱导角膜新生血管模型。碱烧伤后不同时间(1,3,5,7,14d)形态学分析来评价角膜新生血管的情况,免疫组化评价角膜VEGF的表达。结果:角膜碱烧伤后24hVEGF的表达开始升高,伤后5d达高峰(阳性表达最强,遍布全层),伤后7dVEGF的表达开始下降,14d后显著下降。碱烧伤后,角膜新生血管与VEGF的表达呈明显的平行关系。结论:碱烧伤后兔角膜VEGF的表达水平与新生血管的形成有相关性,并在其中发挥了重要作用。     

重组腺相关病毒介导的血管内皮生长因子反义核苷酸对糖尿病视网膜病变的影响

目的观察重组腺相关病毒（rAAV）介导的血管内皮生长因子（VEGF）反义核苷酸（rAAV-aVEGF165）对糖尿病大鼠视网膜VEGF表达的影响。方法 40只Sprague-Dawley大鼠用链尿佐菌素（STZ）腹腔注射诱导糖尿病大鼠模型。去除实验中途死亡和血糖恢复的大鼠，共计32只大鼠纳入研究，随机平均分为实验组和对照组,每组各16只。实验组和对照组大鼠玻璃体腔分别注射rAAV-aVEGF165（10¹⁰ pfu/ml）、磷酸盐缓冲液各10 μl，建模后1、5个月免疫组织化学及Western blot评价视网膜VEGF的表达，并对视网膜血管进行透射电子显微镜观察。结果 1个月时，实验组和对照组大鼠视网膜VEGF表达极低；5个月时，实验组大鼠视网膜VEGF表达较同期对照组降低，差异有统计学意义（t=23.87, P＜0.01）。透射电子显微镜观察显示，实验组视网膜未见明显异常改变，而对照组视网膜微血管腔明显狭窄，基底膜明显增厚，密度不均，并出现断裂，内皮细胞明显肿胀，突起增多，管腔明显狭窄，周细胞内细胞器结构模糊，细胞核固缩，呈凋亡状态，异染色质明显分布不均，浓集。结论 rAAV-aVEGF165能下调视网膜VEGF的表达，从而阻止糖尿病视网膜病变的发生、发展；rAAV是眼反义基因治疗的有效载体。 （中华眼底病杂志，2008，24：255-258）     

视神经疾病165例病因分析

目的 探察视神经病变病因.方法 对同仁眼科中心神经眼科2005年1月至12月门诊随机统计165例视神经疾病患者.结果 眶颅外伤最多见,占18%,其次为Lebet视神经萎缩(LHON)占14.5%,颅内占位性病变占10%,前部缺血性视神经病变(AION)占9%,新生儿缺血缺氧脑病占8%.结论 国人视神经病变仍以眶颅外伤、颅内占位及前部缺血性视神经病变多见,感染性少见,LHON有增多趋势,儿童最常见为新生儿缺血缺氧脑病.     

儿童眼外伤165例分析

目的研究儿童眼外伤的有关特点。方法对165例儿童眼外伤的相关因素进行统计分析。结果儿童眼外伤有其特点:男性儿童多发,眼部伤情重,并发症多,农村多发,致盲率高等。结论儿童眼外伤是主要的致盲原因之一,重在预防。     

Dacryocystorhinostomy. Retrospective study of 165 cases. Indications. Technic. Results. Comparative study of 25 cases of injury with 165 cases including all etiologies

A retrospective study of 165 cases of dacryocystorhinostomy (DCR) has been performed in order to establish a relationship between the results and the delay between the initial symptoms and the DCR. The study compares the results obtained in 25 cases of DCR performed for post traumatic injury to those obtained in 165 cases of DCR performed for various etiologies (traumatic cases included). Traumatic cases are more frequent in men 19/ 25 cases (73%); infectious etiologies are more frequent in women 98/ 118 cases. The results are nearly the same for all the etiologies: 87% good results for the overall (147 cases), 88% good results for the traumatic cases (24 cases). The stenosis occurs within the first month in 72% and before the fourth month in all the cases. There is no difference in the results according to the surgical technique however the small number of each group does not allow a statistical analysis. The results are better when the delay between the initial traumatism or the first surgical repair and the DCR is more than six months (15 good results for 15 cases). The results were worse when the delay is less than 6 months (6 success for 9 cases), with p = 0.07 in the statistical analysis.     

Comparative antiproliferative and cytotoxic profile of bevacizumab (Avastin), pegaptanib (Macugen) and ranibizumab (Lucentis) on different ocular cells

Aim To compare the antiproliferative and cytotoxic properties of bevacizumab (Avastin), pegaptanib (Macugen) and ranibizumab (Lucentis) on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5) and pig choroidal endothelial cells (CEC). Methods Monolayer cultures of ARPE19, RGC5 and CEC were used. Bevacizumab (0.1–0.3 mg/ml), pegaptanib (0.025–0.08 mg/ml) or ranibizumab (0.04–0.125 mg/ml) diluted in culture medium were added to the cells. Expression of VEGF-receptors (VEGFR1 and VEGFR2) and von Willebrand factor (a marker for endothelial cells) were analysed by immunohistochemistry. CEC cells were stimulated with VEGF. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays cells were grown to confluence and then cultured in a serum-depleted medium to ensure a static milieu. MTT-test was performed after one day. Results CEC and ARPE19 cells stained positively for VEGFR1 and VEGFR2. More than 95% of the CEC cells were positive for von Willebrand factor. Ranibizumab reduced CEC cell proliferation by 44.1%, bevacizumab by 38.2% and pegaptanib by 35.1% when the drugs were used at their established clinical doses. The differences, however, between the three drugs in respect to cell growth inhibition were not statistically significant. Only a mild antiproliferative effect of bevacizumab or pegaptanib on ARPE19 cells could be observed. Ranibizumab did not alter ARPE19 cell proliferation. No cytotoxicity on RGC5, CEC and ARPE19 cells could be seen. Conclusions Bevacizumab, pegaptanib and ranibizumab significantly suppress choroidal endothelial cell proliferation. However, when used at the currently established doses none of the drugs was superior over the others in respect to endothelial cell growth inhibition. The biocompatibility of all three drugs — including the off-label bevacizumab — seems to be excellent when used at the currently recommended intravitreal dose. This work is presented on behalf of the Tuebingen Bevacizumab Study Group.     

Intravitreal use of bevacizumab (Avastin) for choroidal neovascularization due to ARMD: a preliminary multifocal-ERG and OCT study

Purpose To evaluate by MFERG and OCT the macular function before and after intravitreal use of bevacizumab (Avastin) in eyes suffering from CNV due to ARMD. Methods Eighteen eyes with subfoveal CNV due to ARMD were studied before and after intravitreal use of bevacizumab with MFERG and OCT. The post treatment follow up was three months. Results Before treatment, OCT shows an increase of the retinal thickening of the fovea and the electrical response densities in the fovea and parafovea were decreased in all patients. Three months after treatment, OCT showed a real resolution of the subretinal fluid. The electrical responses in the fovea and parafovea remained the same or slightly improved in some cases. The intraocular pressure remained normal and no inflammation was observed. Conclusion The intravitreal use of bevacizumab may provide anatomical correlates that support the concept of disease amelioration but the functional improvement of the macula three months after treatment is not obvious. However the method is promising and needs further evaluation.     

The analysis of foreign bodies in the anterior chamber in 165 cases

One hundred and sixty-six eyes of 165 patients with foreign bodies in the anterior chamber who were treated at our center from 1966 to 1988 were retrospectively reviewed. Among them there were 109 eyes with nonmetal foreign bodies, 55 eyes with metal foreign bodies and three eyes with foreign bodies of unknown nature. Most of these foreign bodies were stone or iron. 83.6% of the patients were in the age range from 7 to 40 years, and the left eyes were affected more than the right eyes. Mostly, the foreign bodies were located on the surface of iris (97 cases) and at the anterior chamber angle (40 cases). The rates of blindness were 27.8% and 16.3% before and after operation. The postoperative visual acuity were: 0.05-0.2 in 27 eyes, 0.3-0.9 in 66 eyes, greater than 1.0 in 43 eyes and the rest of the eyes lower than 0.05. The presurgery treatment such as miosis and reduction of intraocular pressure, the selection of operative methods according to the nature, size, location of the foreign bodies and tissue wrapped up were discussed. If combined procedures should be done, it is better to remove the foreign bodies first and then perform the operation for complications.     

Distribution,markers, and functions of retinal microglia

Retinal microglia originate from hemopoietic cells and invade the retina from the retinal margin and the optic disc, most likely via the blood vessels of the ciliary body and iris, and the retinal vasculature, respectively. The microglial precursors that appear in the retina prior to vascularization are major histocompatibility complex (MHC) class I- and II-positive and express the CD 45 marker, but lack specific macrophage markers. They differentiate into ramified parenchymal microglia in the adult retina. A second category of microglial precursors, which do express specific macrophage markers, migrate into the retina along with vascular precursors. They appear around blood vessels in the adult retina and are similar to macrophages or cells of the mononuclear phagocyte series (MPS). Microglia are distributed in the outer plexiform layer (OPL), outer nuclear layer (ONL), inner plexiform layer (IPL), ganglion cell layer (GCL), and nerve fiber layer (NFL) of the primate retina. The pattern of microglial distribution in the avascular retina of the quail indicates that blood vessels are not responsible for the final location of microglia in the retina. In the human retina, microglia express MHC class I, MHC class II, CD 45 , CD68, and S22 markers. In the rat and mouse retina, OX 41 , OX 42 , OX 3 , OX6, OX18, ED1, Mac-1, F 4 /80, 5 D 4 anti-keratan sulfate, and lectins are used to recognize microglia. Microglial cells play an important role in host defense against invading microorganisms, immunoregulation, and tissue repair. During neurodegeneration, activated microglial cells participate in the phagocytosis of debris and facilitate regenerative processes. In autoimmune disease, microglia have dual functions: initiating uveoretinitis, but also limiting subsequent inflammation. Retinal microglia may be associated with vitreoretinopathy, diabetic retinopathy, glaucoma, and age-related macular degeneration. The goal of this article was to review the present knowledge about retinal microglia and the function of retinal microglia in pathological conditions.     

视网膜特异性表达人血管内皮生长因子 基因载体的构建

目的构建在视网膜组织特异性表达的人血管内皮生长因子（VEGF）₁₆₅基因。方法用聚合酶链反应(PCR)方法从BLAB/C鼠全基因组扩增能在视网膜组织特异性表达的rho启动子，经限制性内切酶纯化后克隆于质粒pcDNA^3.1+-VEGF₁₆₅中，建立重组质粒pcDNA^3.1+-rho-VEGF₁₆₅，通过限制性内切酶酶切分析及PCR鉴定筛选出正确重组质粒pcDNA^3.1+-rho-VEGF₁₆₅,由jetPEI介导转染人视网膜色素上皮细胞和人脐静脉内皮细胞，并通过免疫组织化学染色以及绘制细胞生长曲线检测在人视网膜色素上皮细胞和人脐静脉内皮细胞中VEGF蛋白的表达。结果在人视网膜色素上皮细胞中，重组质粒pcDNA^3.1+-rho-VEGF₁₆₅比质粒pcDNA^3.1+-rho-VEGF₁₆₅的VEGF蛋白表达强，在人脐静脉内皮细胞，两者的表达量无明显差别。结论pcDNA^3.1+-rho-VEGF₁₆₅载体的构建为进一步研究VEGF在视网膜新生血管形成中的致病机理提供基础材料，并为进一步建立视网膜特异性表达VEGF转基因鼠模型建立了基础。(中华眼底病杂志,2005,21:106-109)