缺氧对培养的人视网膜色素上皮细胞中NOX4表达的影响

目的:观察缺氧对培养的人视网膜色素上皮细胞中NADPH氧化酶4(NOX4)表达的影响,探讨NADPH氧化酶与脉络膜新生血管(choroidal neovascularization,CNV)形成的关系.方法:人视网膜色素上皮细胞(human retinal pigment epithelium,hRPE)体外常规培养和传代,取3～6代细胞用于实验.将培养的人RPE细胞分为常氧对照组和缺氧处理组,常氧对照组用DMEM完全培养液培养细胞,缺氧处理组培养液中含有终质量浓度为200μmoL/LCoCl2,分别观察缺氧后4、6、8、l2、24h后终止.采用细胞免疫荧光染色技术及Western-blot方法鉴定人RPE细胞中NOX4的表达以及缺氧不同时间对NOX4表达的影响.结果:常氧对照组及缺氧处理组人RPE细胞生长良好,形态呈梭形.缺氧处理组细胞的形态及排列方式与空白对照组相比无明显改变.免疫荧光染色显示,常氧对照组RPE细胞内有少量的NOX4表达,细胞质呈绿色荧光,细胞核呈蓝色荧光;缺氧处理后NOX4的表达量增加,一直持续到缺氧后期;Western-bolt显示缺氧条件下人RPE细胞中NOX4的表达明显增加,与缺氧时间呈正比,与常氧对照组相比,差异具有统计学意义(P＜0.05):结论:NADPH氧化酶在人RPE细胞中有表达,缺氧条件下NOX4的表达显著提高,且与时间呈正比.提示NADPH氧化酶可能在脉络膜新生血管形成中发挥重要调节作用.     

血管内皮生长因子及上皮-间充质转化在增生性玻璃体视网膜病变发病中的作用

增生性玻璃体视网膜病变(PVR)是一种发生在孔源性视网膜脱离(RRD)自然病程中或复位手术后的严重并发症,常常导致患者视力丧失。目前,临床缺乏有效的治疗方法。PVR病理特征是多种细胞在细胞因子的作用下发生的过度炎症反应和异常增生,最终在视网膜表面形成增殖膜及进一步的牵拉性视网膜脱离(TRD)。对PVR发病机制的深入研究将有助于为其治疗寻找有前景的分子靶标。近年研究发现,血管内皮生长因子(VEGF)及视网膜色素上皮(RPE)细胞的上皮-间充质转化(EMT)在PVR发病中发挥着重要作用。本文就VEGF及RPE细胞EMT在PVR发病中的作用,以及二者的相互联动机制进行了总结,以期为PVR的治疗和临床研究提供新的思路。     

上皮-间质转化在视网膜下纤维化发病机制中的研究进展

视网膜下纤维化(SRFi)被定义为视网膜内或视网膜下混合的纤维血管结构,本质是新生血管性年龄相关性黄斑变性(nAMD)中脉络膜新生血管形成后伤口过度愈合的结果,这是导致nAMD患者终末期视力丧失的主要原因。nAMD患者终末期视网膜色素上皮细胞向上皮-间质细胞转化的过程被认为是影响SRFi形成的主要细胞基础。针对SRFi中上皮-间质转化的相关细胞及分子机制研究可能是未来终末期nAMD疾病防治的重要发展方向。     

Optical Coherence Tomography in the Acute and Chronic Phases of Vogt-Koyanagi-Harada Disease

Purpose: To assess the potential of optical coherence tomography (OCT) in the diagnosis and monitoring of serous retinal detachment in Vogt-Koyanagi-Harada (VKH) disease and to describe OCT characteristics of subretinal sequelae of the disease. Methods: Six patients in the acute phase of VKH disease with serous retinal detachment were followed in our department from July 2001 to December 2003 using slit-lamp biomicroscopy, OCT, and fluorescein angiography. Results: OCT was effective in objectively quantifying the amount of serous retinal detachment present and then in following the resolution of subretinal fluid accumulation. Subretinal pigmented lesions on angiography corresponded with retinal pigment epithelium hypertrophy and fibrosis on OCT. Conclusion: A beneficial effect of treatment was observed within days, paralleling the improvement in visual acuity. Retinal pigment epithelium hypertrophy and fibrosis in the chronic phase of the disease were analyzed with OCT for the first time.     

增生性玻璃体视网膜病变中调控上皮间质转化的研究进展

增生性玻璃体视网膜病变是眼外伤和视网膜脱离常见的并发症，可造成视力不可逆转的急剧下降。手术治疗的高复发率促使学者们积极寻求有效的药物辅助治疗方法。虽然该病的发病机制至今仍未完全阐明，但研究表明，视网膜色素上皮细胞经历的上皮-间质转化过程是其发病的关键步骤。近年来学者们对上皮-间质转化在增生性玻璃体视网膜病变中所涉及的信号通路、增殖作用和纤维化、能量代谢和氧化应激、表观遗传以及中药提取物等多方面做了大量研究，本文将对此展开综述。     

Comparison of the rebound tonometer with the Goldmann applanation tonometer in glaucoma patients

PURPOSE: To determine the agreement between the measurement of intraocular pressure (IOP) by the rebound tonometer (RBT) and by the Goldmann applanation tonometer (GAT) and to find out the effect of central corneal thickness (CCT) values on IOP measurements in glaucoma patients. METHODS: IOP was measured with the RBT and GAT, respectively, in 61 eyes of 61 glaucoma patients. CCT was measured using an ultrasonic pachymeter after all IOP determinations had been made. The mean IOP measurement by the RBT was compared with the measurement by the GAT, by Student's t-test. Bland-Altman analysis was performed to assess the clinical agreement between the two methods. The effect of CCT on measured IOP was explored by linear regression analysis. RESULTS: The mean patient age was 56.7+/-21.1 years (range: 30-80 years). There were 32 (52.46%) women and 29 (47.54%) men in the study group. The mean IOP readings were 18.70+/-4.76 mmHg using the RBT, and 18.27+/-3.49 mmHg using the GAT. The difference was not statistically significant (mean difference 0.43+/-2.55, P=0.2). A frequency distribution of the differences demonstrated that in more than 80% of cases the IOP readings differed by <2.3 mmHg between the RBT and GAT. There was a strong correlation between the RBT and GAT readings (r=0.852, P<0.0001). The IOP measurements with the two methods were correlated with CCT (r=0.40, P=0.02 for the RBT and r=0.48, P<0.0001 for the GAT). The IOP increased 1.1 mmHg and 8 mmHg for every 100-microm increase in CCT for the GAT and RBT, respectively. CONCLUSION: The RBT slightly overestimated the IOP value by 0.43 mmHg on average when compared with the GAT. Nevertheless, the RBT readings appeared to be more affected by the various thicknesses of different corneas when compared with those obtained using the GAT.     

上皮-间充质转化在增生性玻璃体视网膜病变发病机制中的作用

增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)是孔源性视网膜脱离及玻璃体视网膜手术后的常见并发症,其主要特征是在视网膜前形成无血管的纤维细胞膜.超微结构和免疫病理研究表明,多种细胞参与PVR的发生与发展,如视网膜色素上皮细胞、神经胶质细胞、巨噬细胞、玻璃体细胞、成纤维细胞和肌成纤维细胞等.其中,肌成纤维细胞是引起PVR增生膜收缩导致视网膜复位手术失败的主要细胞,主要来自于视网膜色素上皮细胞的上皮-间充质转化(epithelial-mesenchymal transition,EMT),在PVR的发生发展中具有重要作用.因此,阻断视网膜色素上皮细胞转分化为肌成纤维细胞可能成为预防和治疗PVR的一种有效的方法.     

Electro-oculogram in patients with neurofibromatosis type 1

Purpose: The electro-oculogram (EOG) is a powerful test to evaluate the functional status of the retinal pigment epithelium (RPE). Clinically detectable changes of the RPE desribed in neurofibromatosis type 1 (NF-1) patients include combined hamartoma of the retina/RPE and congenital hypertrophy of the RPE. The goal of this study was to determine whether the function of RPE as measured by EOG is also changed in individuals with NF-1. Patients: Studies were undertaken in 20 patients with clinically diagnosed NF-1 and compared to 16 normal healthy controls. Methods: Standard EOG and flash ERG recordings were performed in accordance with International Society for Clinical Electrophysiology of Vision (ISCEV) standards. Results: In NF-1 patients the Arden indexes of the EOG test were significantly higher primarily due to the lower values of dark troughs. Supernormal EOGs were present in 60% of NF-1 patients in comparison to the control group mean +2 SD. No one patient showed so high abnormalities during flash ERG examination. Conclusions: Dysfunction of the RPE may be characteristic feature of individuals with NF-1.     

Role of glycolysis in retinal vascular endothelium, glia, pigment epithelium, and photoreceptor cells and as therapeutic targets for related retinal diseases

Glycolysis produces large amounts of adenosine triphosphate (ATP) in a short time. The retinal vascular endothelium feeds itself primarily through aerobic glycolysis with less ATP. But when it generates new vessels, aerobic glycolysis provides rapid and abundant ATP support for angiogenesis, and thus inhibition of glycolysis in endothelial cells can be a target for the treatment of neovascularization. Aerobic glycolysis has a protective effect on Müller cells, and it can provide with a target for visual protection and maintenance of the blood-retinal barrier. Under physiological conditions, the mitochondria of RPE can use lactic acid produced by photoreceptor cells as an energy source to provide ATP for survival. In pathological conditions, because RPE cells avoid their oxidative damage by increasing glycolysis, a large number of glycolysis products accumulate, which in turn has a toxic effect on photoreceptor cells. This shows that stabilizing the function of RPE mitochondria may become a target for the treatment of diseases such as retinal degeneration. The decrease of aerobic glycolysis leads to the decline of photoreceptor cell function and impaired vision; therefore, aerobic glycolysis of stable photoreceptor cells provides a reliable target for delaying vision loss. It is of great significance to study the role of glycolysis in various retinal cells for the targeted treatment of ocular fundus diseases.     

活性氧在贝伐单抗诱导视网膜色素上皮细胞上皮间质转化中的作用

目的　观察贝伐单抗（BEV）对人视网膜色素上皮（RPE）细胞氧化应激损伤和上皮间质转化（EMT）的影响,进一步探讨抑制活性氧（ROS）在EMT中的作用。方法　选取人ARPE-19细胞株,根据实验需要将ARPE-19细胞分为4组:空白对照组、BEV组、BEV+NAC组和BEV+DPI组,其中空白对照组细胞不进行任何干预,BEV组用0.25 g?L^-1 BEV处理细胞72 h,另外两组在 BEV处理细胞24 h后分别再用ROS抑制剂NAC和NADPH氧化酶抑制剂DPI处理细胞48 h。采用2,7-二氯二氢荧光素二乙酸酯（DCFH-DA）染色观察各组ARPE-19细胞内ROS和H₂O₂的生成堆积情况;细胞免疫荧光观察各组ARPE-19细胞中EMT标志物［紧密连接相关蛋白闭锁带蛋白-1（ZO-1）、α-平滑肌肌动蛋白（α-SMA）和纤维连接蛋白（FN）］的表达情况;再通过RT-PCR和Western blot检测各组细胞中EMT标志物的mRNA和蛋白的表达水平。结果　DCFH-DA染色观察发现,ARPE-19细胞加入BEV继续培养后,ROS和H₂O₂表达均明显上调（均为P＜0.05）。与空白对照组相比,BEV组EMT指标变化显著:上皮标志物ZO-1的表达降低,间质标志物α-SMA和FN的表达升高,两组间各指标相比差异均有统计学意义（均为P＜0.05）;与BEV组相比,BEV+NAC组和BEV+DPI组中各项指标mRNA表达变化显著,ZO-1上升至0.955±0.048、1.056±0.017,而α-SMA下降至0.982±0.165、1.058±0.165,此外FN表达（0.666±0.063、0.983±0.125）也显著降低,差异均有统计学意义（均为P＜0.05）。各种因子的蛋白表达变化趋势与其相应mRNA表达相一致,与单纯BEV组相比,ROS抑制剂NAC和NADPH氧化酶抑制剂DPI都显著改变了EMT标志物的表达,ZO-1蛋白相对表达量升高了0.195±0.010、0.770±0.175,而α-SMA下降了0.353±0.098、0.482±0.037,FN下降了0.528±0.161、0.612±0.134,差异均有统计学意义（均为P＜0.05）。结论　ROS参与了BEV诱导的RPE细胞EMT,抑制ROS可减轻BEV诱导的人RPE细胞的 EMT程度。     

Role of 4-hydroxynonenal in epidermal growth factor receptor-mediated signaling in retinal pigment epithelial cells

Lipid peroxidation (LPO) end-product 4-hydroxynonenal (4-HNE) has been implicated in the mechanism of retinopathy. Lately it has been shown that besides being cytotoxic, 4-HNE plays an important role in oxidative stress-induced signaling. In this study, we have investigated the effect of 4-HNE on epidermal growth factor receptor (EGFR)-mediated signaling, its potential functional consequences, and the regulatory role of the 4-HNE metabolizing isozymes, glutathione S-transferase A4-4 (GSTA4-4) on this signaling in retinal pigment epithelial (RPE) cells. Our results showed that consistent with its known toxicity at relatively higher concentrations, 4-HNE induced cell death in RPE. However, at lower concentrations (as low as 0.1 μM) 4-HNE triggered phosphorylation of EGFR and activation of its down stream signaling components ERK1/2 and Akt that are known to be involved in cell proliferation. These effects of 4-HNE on EGFR could be attenuated by the over expression of GSTA4-4 that reduces intracellular levels of 4-HNE. Our results also indicated that 4-HNE-induced activation of EGFR is a protective mechanism against oxidative stress because EGFR, MEK, and PI3K inhibitors potentiated the toxicity of 4-HNE and also inhibited wound healing in a RPE cell model. These studies suggest that as an initial response to oxidative stress, 4-HNE induces protective mechanism(s) in RPE cells through EGFR-mediated signaling.     

细胞外基质在年龄相关性黄斑变性中的研究进展

年龄相关性黄斑变性(ARMD)是老年人失明的主要原因。研究表明,细胞外基质(ECM)调节障碍作为ARMD疾病的重要特征之一,它的损伤可贯穿于ARMD病程。此外,参与ARMD发生发展过程的各类型细胞可以在多种信号的控制下参与ECM的形成与异常沉积,通过传递调节黏附、迁移、增殖、凋亡、存活或分化的信号,从而导致视网膜、脉络膜微环境的破坏,免疫功能障碍,浸润性炎症细胞分化,新生血管生成,上皮-间充质转化,最终导致晚期ARMD视网膜下纤维化和瘢痕形成及视力严重受损。因此,ECM在ARMD中的作用逐渐引起重视。文章针对视网膜中ECM与ARMD的联系及ARMD中各类型细胞与ECM之间的作用做一综述,以期为治疗ARMD的研究方向提供指导意义。     

Detection of Mycobacterium tuberculosis Genome in Subretinal Fluid of Patients with Latent Tuberculosis Infection

Purpose: To test our hypothesis that Mycobacterium tuberculosis (MTB) DNA may be present in retinal pigment epithelium (RPE) cells in individuals with latent tuberculosis (TB) infection.

Methods: A total of 100 patients with rhegmatogenous retinal detachment (without any signs of endogenous uveitis in either eye) underwent pars plana vitrectomy (PPV) with internal tamponade. All were subjected to tuberculin skin test. None had manifest systemic TB disease. The subretinal fluid collected during surgery was subjected to multitargeted polymerase chain reaction (PCR) targeting three genes of MTB (IS6110, MPB64 and protein b).

Results: In total, 16 patients had latent TB, of which TB PCR was positive in six patients.

Conclusions: We demonstrate presence of MTB genome in the subretinal fluid containing RPE cells from individuals with latent TB infection, who did not have any evidence of intraocular TB or manifest systemic TB disease, and suggest that MTB can be sequestrated in the RPE cells in latent TB.     

藤黄酸通过CYLD/NF-κB信号通路抑制高糖环境下RPE细胞上皮-间充质转化的作用

目的 基于去泛素化酶圆柱瘤蛋白/核因子κB(CYLD/NF-κB)通路,研究藤黄酸(GA)对高糖环境下视网膜色素上皮(RPE)细胞上皮-间充质转化(EMT)的作用。方法 体外培养ARPE-19细胞,高糖诱导,实验分为NC组(含5.5 mmol·L^-1葡萄糖),HG组(含30 mmol·L^-1葡萄糖),HG+不同剂量GA组(2μmol·L^-1、4μmol·L^-1、8μmol·L^-1GA分别预处理RPE细胞1 h,加30 mmol·L^-1葡萄糖)。CCK-8检测各组RPE细胞增殖活力,免疫荧光染色检测RPE细胞中α平滑肌肌动蛋白(α-SMA)、去泛素化酶圆柱瘤蛋白(CYLD)表达;Western blot检测RPE细胞中α-SMA、CYLD、磷酸化核转录因子κB(p-NF-κB)蛋白表达。结果 与NC组相比,HG组RPE细胞出现明显增殖,RPE细胞中α-SMA、p-NF-κB蛋白表达均增加,CYLD蛋白表达减少(均为P<0.01);与HG组相比,HG+不...     

诱导多能干细胞治疗年龄相关性黄斑变性的研究进展

年龄相关性黄斑变性是造成中老年人失明的主要原因,严重影响了患者的生活质量,但目前尚无有效疗法。随着病情的进展,视网膜色素上皮层细胞逐渐退化,并最终引起视力的不可逆性损害。诱导多能干细胞的出现提供了可供移植的视网膜色素上皮细胞来源,避免了免疫及伦理等问题,但仍然需要克服许多障碍和困难。     

蓝光诱导视网膜色素上皮细胞铁死亡的机制研究

目的:探讨蓝光诱导人视网膜色素上皮(ARPE)细胞铁死亡的发生及可能机制。方法:体外培养的ARPE-19细胞接受405nm蓝光50mW/cm²辐照度照射不同时间,分为对照组、16.3J/cm²组、32.6J/cm²组和65.2J/cm²组;将65.2J/cm²组定为高能量蓝光照射组,进一步分为对照组、高能量蓝光照射组和高能量蓝光照射+铁死亡抑制剂组,CCK-8检测细胞活力,试剂盒检测细胞内谷胱甘肽(GSH)含量、二价铁离子浓度及丙二醛(MDA)含量,Western blot法检测细胞内GPX4和xCT蛋白相对表达量。结果:蓝光照射导致ARPE-19细胞活力下降呈剂量依赖性,高能量蓝光照射导致细胞内GSH含量下降,二价铁离子浓度和MDA含量上升(均P<0.05);加入铁死亡抑制剂可部分恢复蓝光照射组细胞活力和GSH含量,减少MDA含量,降低二价铁离子浓度(均P<0.05);蓝光照射组GPX4和xCT蛋白相对表达量显著下降,加入铁死亡抑制剂后蛋白表达量不同程度恢复(P&...     

微小RNA调控增生性玻璃体视网膜病变的研究进展

增生性玻璃体视网膜病变(PVR)是一个眼部组织的创伤修复和纤维化过程,其特征性改变是在玻璃体腔和(或)视网膜表面形成由细胞外基质(ECM)和各种类型的细胞组成的视网膜前膜(ERM),ERM收缩形成视网膜皱褶,并牵拉视网膜引起视网膜脱离(RD)。视网膜色素上皮(RPE)细胞发生上皮-间质转化(EMT)和ECM累积是ERM形成的重要病理机制。转化生长因子-β(TGF-β)等诱导RPE细胞发生EMT,细胞失去上皮表型、细胞间黏附减弱和表达间充质表型。由间充质细胞分化而成的成纤维细胞样细胞分泌ECM等成分,与神经胶质细胞、成纤维细胞等共同形成ERM。RPE细胞的EMT过程受许多细胞因子/生长因子、转录因子及微小RNA(microRNA,miRNA)等的调控,研究证明miRNA是一类新型且功能强大的调节基因,在PVR的EMT过程中起着重要调控作用。本文将近年来miRNA调控PVR的作用机制和干预性治疗研究进行综述。     

结缔组织生长因子在人视网膜色素上皮细胞的增生、迁移和上皮细胞-间充质细胞转变中的作用

目的　研究结缔组织生长因子（ｃｏｎｎｅｃｔｉｖｅｔｉｓｓｕｅｇｒｏｗｔｈｆａｃｔｏｒ,ＣＴＧＦ）在人视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ,ＲＰＥ）细胞的增生、迁移和上皮细胞-间充质细胞转变（ｅｐｉｔｈｅｌｉａｌｍｅｓｅｎｃｈｙｍａｌｔｒａｎｓｉｔｉｏｎ,ＥＭＴ）中的作用。方法　采用ＣＣＫ-８细胞计数试剂盒测定４００ｎｇ?ｍＬ－１ＣＴＧＦ处理后的ＡＲＰＥ-１９细胞以及稳定表达ＣＴＧＦｓｉＲＮＡ或对照ｓｉＲＮＡ的ＡＲＰＥ-１９细胞的增生情况。采用细胞划痕实验评估ＣＴＧＦＲＮＡｉ对ＡＲＰＥ-１９细胞迁移的影响,同时测定稳定表达ＣＴＧＦｓｉＲＮＡ或对照ｓｉＲＮＡ的ＡＲＰＥ-１９细胞中ＣＴＧＦ、ＦＮ、ＭＭＰ-２和α-ＳＭＡ的表达水平以评估ＣＴＧＦ对于ＴＧＦβ１诱导的ＥＭＴ作用。结果　４００ｎｇ?ｍＬ－１ＣＴＧＦ处理组ＡＰＲＥ-１９细胞与未接受ＣＴＧＦ处理组细胞之间以及ＣＴＧＦｓｉＲＮＡ处理组与阴性对照ｓｉＲＮＡ组之间的细胞倍增时间均存在显著差异（均为Ｐ＜０．０５）。正常培养ＡＲＰＥ-１９细胞组修复划痕的时间（≤４８ｈ）显著少于ＣＴＧＦＲＮＡｉ靶向干扰处理组（≥７２ｈ）。以ＴＧＦ-β１诱导ＥＭＴ,ＣＴＧＦｓｉＲＮＡ处理组与阴性对照组比较,ＣＴＧＦ、α-ＳＭＡ、ＦＮ和ＭＭＰ-２表达均显著下调。此外,外源性ＣＴＧＦ可单独诱导ＡＲＰＥ-１９细胞α-ＳＭＡ表达增加。结论　ＣＴＧＦ能够促进ＡＲＰＥ-１９细胞增生,而ＣＴＧＦＲＮＡｉ不但能够显著抑制其增生还可显著抑制由划痕实验引发的ＡＲＰＥ-１９细胞的迁移。此外,ＣＴＧＦ可通过上调α-ＳＭＡ表达水平而增强ＡＲＰＥ-１９细胞对于ＴＧＦ-β１的反应,ＣＴＧＦＲＮＡｉ可使ＴＧＦ-β１诱导的ＥＭＴ减弱。     

Comparison of serum glycosylated hemoglobin levels in patients with diabetic cystoid macular edema with and without serous macular detachment

Aim:

A clinical comparative trial was conducted to compare the levels of glycosylated hemoglobin (HbA1c) in patients with diabetic cystoid macular edema (CME) with and without serous macula detachment (SMD).

Materials and Methods:

Thirty patients (group 1) with diabetic CME in both eyes, but without SMD, and 30 patients (group 2) with diabetic CME and SMD in both eyes documented by optical coherence tomography (OCT) and fundus fluorescein angiography (FFA), were included in the study. In addition to the measurement of central macular thickness by OCT and visual acuity (VA) (as logMAR) using the the early treatment diabetic retinopathy study (ETDRS) chart, the concentrations of HbA1c were measured by high performance liquid chromatography (HPLC). Statistical analysis was done by independent samples t test.

Results:

The mean logMAR VA was 0.8 ± 0.22 (1.0–0.5) in group 1and 0.7 ± 0.16 (1.0–0.6) in group 2. The mean central macular thickness, as determined by OCT, was 468.70 ± 70.44 μm (344–602 μm) in group 1 and 477.80 ± 73.34 μm (354–612 μm) in group 2. The difference between the groups was not statistically significant (P = 0.626). The mean HbA1c levels were 8.16 ± 0.99% in group 1 and 10.05 ± 1.66% in group 2. The difference between the groups was statistically significant (P < 0.001).

Conclusions:

The presence of SMD and high HbA1c levels in the patients with diabetic CME may be indirectly suggestive of retinal pigment epithelium dysfunction.