视网膜细胞凋亡及其调控的研究进展

细胞凋亡与视网膜变性疾病、视网膜脱离和青光眼等多种眼病中视网膜光感受器细胞和神经节细胞等的死亡有密切关系,是视功能丧失的主要原因。神经营养因子通过调控凋亡通路,从而对视网膜细胞产生保护作用。我们综述了视网膜细胞凋亡及其调控以及神经营养因子对凋亡调控的影响在视功能丧失方面的研究进展。     

凋亡相关信号通路在早期糖尿病视网膜病变中的研究进展

视网膜细胞凋亡是早期糖尿病视网膜病变(diabetic retinopathy,
DR)的病理特征,而凋亡相关信号通路在此过程中起了关键作用。早期干预视网膜细胞凋亡的相关信号通路,保护视网膜细胞,对保存DR患者视功能和延缓DR的进展可能起重要作用。本文就迄今较为明确的与DR凋亡相关的信号通路,即线粒体通路、死亡受体通路、内质网应激通路等在早期糖尿病视网膜病变中的作用及研究进展作一综述。     

外源性促红细胞生成素对大鼠视网膜脱离光感受器细胞的保护作用

目的 观察外源性促红细胞生成素(EPO)对大鼠视网膜脱离(RD)状态下光感受器细胞的保护作用.方法 采用计算机产生随机数字的方法将162只正常雄性SD大鼠分为正常对照组(NC组)、RD组、RD+磷酸盐缓冲液(PBS)组、RD+EPO 100、200、400 ng组.RD后3 d,采用蛋白免疫印迹(Western blot)检测半胱氨酸蛋白酶3(caspase-3)活性和B细胞淋巴瘤/白血病-XL(bcl-XL)的表达,免疫荧光检测caspase-3活性,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测光感受器细胞凋亡情况.RD后14、28 d,2个月,视网膜组织病理学观察并测量外核层(ONL)厚度.结果 Western bolt检测发现,各组间活化caspase-3条带灰度值差异有统计学意义(F=35.96,P<0.01),bcl-XL条带灰度值差异也有统计学意义(F=30.75,P<0.01).免疫荧光和TUNEL检测发现,caspase-3免疫阳性光感受器细胞数目与TUNEL阳性细胞核趋势表现一致.RD后14 d、2个月,各组间ONL差异有统计学意义(F=21.52,96.25;P<0.01).结论 RD后补充外源性EPO可通过抑制caspase-3活性并增加bcl-XL的表达拮抗凋亡,从而发挥对光感受器细胞的保护作用.     

光感受器与双极细胞间突触在视网膜脱离后的重塑

视网膜脱离（RD）是严重的致盲性眼病。近年来研究发现,RD后光感受器与双极细胞间突触结构会遭到破坏,这一组织学上的变化对RD患者术后视功能的恢复有着重要影响。现就光感受器与双极细胞间突触在RD后的重塑进行综述,探讨RD术后视功能恢复不良的可能机制。     

视网膜细胞凋亡与bcl-2基因

细胞凋亡与视网膜光损伤、视网膜变性、青光眼等多种眼病中视网膜光感受器细胞、神经节细胞等的死亡有密切关系,是视功能丧失的主要原因。凋亡调控基因bcl-2家族成员可分为凋亡阻遏基因(bcl-2、bcl-XL、mcl-1、bag-1、a1等)和凋亡促进基因(bax、bak、bcl-Xs、bik、bad等),这些基因编码的蛋白质分子通过组成和/或影响同二聚体与异二聚体的不同比例而介导其对细胞存活的生物学效应。本文就一些伴有视网膜细胞凋亡的常见眼病所致bcl-2基因表达的变化及其相关机制作一综述。     

视网膜神经节细胞凋亡的信号传导与基因调控

青光眼视功能损害的病理基础是视网膜神经节细胞（RGCs)的进行性死亡和视神经纤维的丢失，神经节细胞死亡主要是通过细胞凋亡的方式进行的。刺激RGCs凋亡的信号有多种，如视网膜缺血，兴奋性谷氨酸释放，一氧化氮（NO）增多，神经营养因子剥夺及其他细胞外环境的改变。这些细胞外部因素通过不同的信号传导途径影响着细胞内控制凋亡的相关基因，如caspase-3,bcl-2及p53，从而间接地调控细胞凋亡。从视网膜神经节细胞凋亡的信号传导途径和细胞凋亡的基因调控两方面介绍其研究进展。并探求其可能的保护机制。     

实验性视网膜脱离时外源性bFGF对视网膜细胞凋亡的干预作用

目的 研究实验性视网膜脱离(RD)后家兔视网膜细胞凋亡情况,探讨RD后视功能损伤机制,观察碱性成纤维细胞生长因子(bFGF)对细胞凋亡的干预作用.方法 从42只青紫兰兔中随机选取40只兔,将每只兔的左、右眼分为实验对照组和治疗组.视网膜下注射玻璃酸钠(爱维)建立RD动物模型后,取右眼在玻璃体腔内注射10IU/20μl bFGF溶液,作为治疗组;左眼注射平衡盐溶液(BSS)20μl作为实验对照组.剩余2只家兔设为正常对照组,不作任何处理,在实验观察结束时取眼球.三组均采用HE染色、原位末端标记法(TUNEL)和透射电子显微镜观察视网膜细胞凋亡情况,并进行细胞计数和统计学检验.结果 RD早期就有细胞凋亡的发生.视网膜各层细胞均可见凋亡细胞,内核层、节细胞层分布最多.凋亡细胞数随脱离时间延长而变化(P<0.01).bFGF组的凋亡细胞数比BSS组少(P<0.01).电镜下可见除正常对照组基本无凋亡细胞外,其余两组均观察到凋亡细胞,.bFGF组中典型凋亡细胞明显少于实验对照组.结论 实验性RD时视网膜组织细胞中存在异常凋亡,可能是RD后视功能损伤机制之一.玻璃体腔内给予bFGF可以抑制视网膜细胞凋亡,为临床治疗视网膜脱离提供参考.     

视网膜脱离视细胞凋亡及其抗凋亡治疗研究进展

视网膜脱离是主要的致盲眼病之一,其发生机制纷繁复杂.目前研究认为光感受器等视细胞的凋亡在视网膜脱离预后中起重要作用.视网膜脱离视细胞凋亡的机制逐步被阐明,相关的光感受器细胞抗凋亡治疗研究也逐渐成为治疗视网膜脱离的热点.光感受器细胞的抗凋亡治疗结合现代视网膜脱离手术将是未来治疗视网膜脱离的理想模式.     

调节性细胞死亡在糖尿病视网膜病变中的研究进展

调节性细胞死亡(RCD)是一种独特的细胞死亡方式,包括细胞凋亡等多种形式。RCD参与人类正常生理活动的诸多环节,更与多种疾病的发生发展密切相关。糖尿病视网膜病变(DR)是近年来发病率较高的一种致盲性眼病。研究表明,DR的发生与细胞凋亡、铁死亡、细胞焦亡、细胞自噬、依赖性细胞死亡等多种RCD的相关蛋白异常表达有关。本文将对DR发病过程中与RCD相关的机制作一综述,以期为DR的靶向治疗提供思路。     

神经营养因子治疗视网膜色素变性的研究进展

目前为止发现了30余种视网膜色素变性（RP）的致病相关基因,发病机制研究提示凋亡可能是它们引起光感受器细胞萎缩的共同病理途径.神经营养因子是一类对神经系统的分化、发育,对神经元的存活,轴突再生均有重要作用的细胞因子.它们可能通过调控视网膜光感受器细胞的凋亡过程起到神经保护作用,有望成为治疗RP的有效药物.本文对近年来神经营养因子的光感受器保护作用、给药途径、治疗机制、副作用等方面的研究状况进行综述,其中基因工程改造的神经营养因子和基因修饰细胞半透膜埋植系统的研究取得了进展.     

Photoreceptor cell death and rescue in retinal detachment and degenerations

Photoreceptor cell death is the ultimate cause of vision loss in various retinal disorders, including retinal detachment (RD). Photoreceptor cell death has been thought to occur mainly through apoptosis, which is the most characterized form of programmed cell death. The caspase family of cysteine proteases plays a central role for inducing apoptosis, and in experimental models of RD, dying photoreceptor cells exhibit caspase activation; however, there is a paradox that caspase inhibition alone does not provide a sufficient protection against photoreceptor cell loss, suggesting that other mechanisms of cell death are involved. Recent accumulating evidence demonstrates that non-apoptotic forms of cell death, such as autophagy and necrosis, are also regulated by specific molecular machinery, such as those mediated by autophagy-related proteins and receptor-interacting protein kinases, respectively. Here we summarize the current knowledge of cell death signaling and its roles in photoreceptor cell death after RD and other retinal degenerative diseases. A body of studies indicate that not only apoptotic but also autophagic and necrotic signaling are involved in photoreceptor cell death, and that combined targeting of these pathways may be an effective neuroprotective strategy for retinal diseases associated with photoreceptor cell loss.     

Apoptosis and other cell death mechanisms after retinal detachment: implications for photoreceptor rescue

Retinal detachment (RD) is one of the most common causes of blindness. This separation of the neurosensory retina from its underlying retinal pigment epithelium results in photoreceptor loss, which is the basis of permanent visual impairment. This review explores the various cell death mechanisms in photoreceptor death associated with RD. One of the major mechanisms is apoptosis, mediated by the intrinsic pathway, the Fas signalling pathway and/or the caspase-independent pathway. Other pathways of mechanisms include endoplasmic reticulum stress-mediated cell death, programmed necrosis and cytokine-related pathways. Understanding the mechanism of RD-associated photoreceptor death is likely to help us improve the current therapies or devise new strategies for this sight-threatening condition.     

Preventing retinal detachment-associated photoreceptor cell loss in Bax-deficient mice

PURPOSE: To characterize photoreceptor cell apoptosis and cell loss in a mouse model of experimental retinal detachment (RD), and to use the technology of mouse genetics to study the molecular mechanisms underlying RD-associated photoreceptor degeneration. METHODS: Retinal detachments were created in adult wild-type and Bax-deficient mice by subretinal injection of 1.4% sodium hyaluronate. At 1, 3, 7, and 28 days after injection, animals were killed, eyes enucleated, and retinal sections studied by histochemistry, immunofluorescence labeling, and confocal microscopy. Rods and cones were labeled, and apoptotic cells were identified with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Photoreceptor cell apoptosis and cell loss were assessed quantitatively by counting both surviving and TUNEL-positive rods and cones. RESULTS: TUNEL-positive cells were found within the outer nuclear layer (ONL) of the detached portions of the retina. They were detected in the detached retina on day 1, peaked on day 3, and dropped precipitously after day 7 after RD. Photoreceptor cell loss of both rods and cones followed a similar time course after RD. Moreover, deletion of the proapoptotic gene Bax in a knockout mouse model abolished the RD-associated photoreceptor cell degeneration. CONCLUSIONS: Apoptosis is a major mechanism leading to photoreceptor cell death after RD. Blockage of the activity of the proapoptotic molecule Bax in a knockout mouse model prevents photoreceptor cell apoptosis and cell loss. These data suggest that the Bax-mediated apoptotic signaling pathway plays a critical role in RD-associated photoreceptor cell death.     

Photoreceptor apoptosis in human retinal detachment

PURPOSE: Although photoreceptor cell apoptosis has been demonstrated in various animal models of retinal detachment (RD), little is known about its occurrence in human RD. We sought to determine whether human photoreceptor cells undergo apoptosis in response to primary and recurrent RD. DESIGN: Prospective, clinical-pathologic, case series. METHODS: Retinal tissue fragments excised during the course of vitreous surgery for RD and recurrent RD were frozen, cut into 4-mum sections, and analyzed using a TdT-dUTP terminal nick-end labeling assay for cell apoptosis. The onset of patient symptoms was used to estimate the duration of the RD. RESULTS: There were eight patients with primary RD and four patients with recurrent RD enrolled in this study. Duration of RD ranged from 1 to 180 days in the primary RD group, and 2 to 15 days in the recurrent RD group. All retinal tissue specimens had TUNEL-positive cells localized to the outer nuclear layer of the retina, consistent with the localization of photoreceptor cell bodies. TUNEL-positive cells were first identified at 24 hours, peaked by 2 days, and dropped to a low level by 7 days after RD. Recurrent RD induced a greater number of TUNEL-positive cells/mm(2) in the ONL compared with primary RD at corresponding timepoints after the onset of RD. CONCLUSIONS: In response to primary and recurrent RD, human photoreceptor cells follow a pattern of apoptosis that is similar to that seen in animal models of RD. This study suggests that photoreceptor cell apoptosis may be one of the causes of reduced vision after RD, especially those that involve the macula. Drugs that inhibit photoreceptor apoptosis may help improve the final visual prognosis of patients with RD.     

Critical role of photoreceptor apoptosis in functional damage after retinal detachment

PURPOSE: Although apoptosis is assumed to play a pivotal role in retinal function loss, its mechanism and real influence on retinal function are still unclear. To investigate the relation between retinal function and apoptosis, we studied photoreceptor apoptosis in experimental retinal detachment (RD). METHODS: We induced RD by subretinal injection of sodium hyaluronate in Brown Norway rats. Apoptotic photoreceptors were detected by TdT-dUTP Terminal Nick-End Labeling (TUNEL). To evaluate the function of the detached retina, electroretinograms (ERGs) were taken on day 1, 3 with corneal electrodes and full-field stimulation. RESULTS: Apoptotic DNA fragmentation appeared 12 hours after RD, was most prominent on day 3, and decreased thereafter. The ERGs showed that the amplitudes of dark-adapted a-waves and light adapted 2 Hz b-waves decreased immediately after RD and continued to decrease over time. The administration of Fas/Fc chimera recombinant protein or a caspase inhibitor, Z-VAD.fmk, failed to prevent either photoreceptor apoptosis or retinal functional damage. In contrast, brain derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) significantly impeded both apoptosis and dysfunction. The ERGs recognized the functional changes sensitively, and these ERG changes correlated well to the amount of photoreceptor apoptosis. Immunohistochemical study showed that apoptosis-inducing factor (AIF), a novel caspase-independent apoptotic factor, was relocalized from mitochondria to the nucleus in this process. CONCLUSIONS: The present results showed that apoptosis was a key phenomenon in the retinal dysfunction in RD and that this process was transmitted mainly by mitochondria-dependent pathways rather than Fas/Fas-L or downstream caspase dependent pathways.     

Photoreceptor decay over time and apoptosis in experimental retinal detachment

Background: Data are scarce on the actual rate and mode of outer nuclear layer decay in retinal detachment (RD). We used an experimental rabbit model to assess the presence of apoptosis and rate of photoreceptor death following RD. This model included the creation of localized and stable retinal blebs, while controlling for any decline of retinal elevation over time.

Methods: RD was produced in New Zealand white rabbits by injecting 0.05 ml of 15% sodium hyaluronate (Healon GV) under the neural retina using a microsurgical technique. Animals were killed at 1, 2, 4, 7, 14 and 29 days. Retinal tissue was processed for light and electron microscopy and for in situ end labeling of fragmented DNA using a modification of the TUNEL technique. Photoreceptor cell nuclei were counted in the RD areas of maximum retinal elevation of 28 eyes, and an additional 4 eyes were used for nick end labeling.

Results: Positive DNA nick end labeling, ultrastructural features and absence of necrotic cells indicated apoptotic photoreceptor cell death. Also, there was a rapid, almost linear elimination of photoreceptor nuclei over time. At 14 days only half of the number of nuclei were discernible, while approximately one tenth remained after 29 days. There was a statistically significant, but minimal decline in RD height over the 4 weeks of study.

Conclusion: Following experimental RD in rabbits, apoptotic cell death is associated with an almost linear elimination of photoreceptor cells over time. The use of highly viscous sodium hyaluronate in separating the neural retina from the retinal pigment epithelium allows the RD to maintain a nearly constant height over a period of 4 weeks.

     

兔视网膜脱离后出现神经感觉层细胞凋亡

目的:研究实验性视网膜脱离(retinal detachment,RD)及脱离复位后神经感觉层细胞凋亡情况,探讨RD后视功能损害的机制为临床治疗提供理论基础。方法:健康成年无眼部疾病青紫兰兔40只,采用计算机随机数字表法分为RD后1,3h;1,3,7,14,28d组及正常对照组,共8组,每组5只,选取右眼为致伤眼,视网膜下注射透明质酸钠建立RD模型;分别于建立模型后按时获取兔眼标本,以TUNEL法观察视网膜神经感觉层细胞的凋亡情况。结果:视网膜脱离复位后存在着神经感觉层细胞凋亡现象,正常对照组、14d和28d组几乎不见凋亡细胞,脱离后1,3h;3,7d组在视网膜各细胞层均出现较多的、具有凋亡形态学与生化改变特征的凋亡细胞,其中在3d组视网膜感觉层细胞凋亡细胞数量达到高峰。视网膜神经感觉层凋亡细胞数目在1,3h;3,7d组之间两两比较均有显著性差异(P<0.01);1,3h;3,7d组与正常对照组、1h;1,28d组之间两两比较均有显著性差异(P<0.01);正常对照组、1h;14,28d组之间两两比较无显著性差异。结论:RD复位后,神经感觉层细胞发生凋亡。     

Apoptosis in proliferative vitreoretinopathy

PURPOSE: To study the involvement of apoptosis using different apoptosis markers in PVR pathogenesis. METHODS: The presence of mRNA coding for Fas, Fas ligand (FasL), and TNF-related apoptosis inducing ligand (TRAIL) was investigated in vitreous samples from 46 consecutive patients-25 with PVR, 11 with retinal detachment (RD) not complicated by PVR, and 10 with macular hole (MH)-using RT-PCR. From previously examined vitreous samples, 21 PVR, 9 RD, and 10 MH were examined for their levels of TGF-beta2 protein with sandwich ELISA kits. Five epiretinal membranes excised from five patients with PVR were also examined for apoptotic cell death using the terminal deoxytransferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) technique. RESULTS: FAS mRNA was detected in 72% of patients with PVR, 55% of patients with RD and 20% of patients with MH. TRAIL mRNA was detected in 67% of patients with PVR, 89% of patients with RD, and 20% of patients with MH. FasL mRNA was detected in 20% of patients with PVR, 9% of patients with RD, and 10% of patients with MH. The median levels of Fas and TRAIL mRNA were significantly higher (P < 0.05) in patients with PVR than in those with MH hole but between patients with PVR and those with RD the difference was not significant (P > 0.05). A significant difference was detected between RD and MH for TRAIL mRNA levels (P = 0.008). For FasL, no significant difference between groups was found. TGF-beta2 was detected in all investigated vitreous samples. A significant difference was found between the PVR and MH groups (P = 0.001) and between the RD and MH groups (P = 0.004), but not between the PVR and RD groups (P < 0.05). The level of TGF-beta2 was significantly correlated to the level of TRAIL mRNA (r = 0.86), but no correlation was found between TGF-beta2 and Fas mRNA levels (r = 0.21). Four of five examined PVR epiretinal membranes showed positive staining for apoptotic cells using the TUNEL technique. CONCLUSIONS: Apoptosis is one of the mechanisms that is involved in PVR pathogenesis. Different apoptosis markers suggest different pathways occur in PVR, including Fas/FasL, TRAIL, and TGF-beta2 mediated processes.     

Prediction of visual outcome after retinal detachment surgery using the Lotmar visometer

AIM: To evaluate whether an achromatic interferometer, the Lotmar visometer, is useful in predicting postoperative visual outcome in patients with primary rhegmatogenous retinal detachment (RD) involving the macula. METHODS: This prospective study included 40 eyes of 40 non-consecutive patients with macula-off RD. The eyes were phakic or pseudophakic, had a clear optical media, and had a measurable potential vision on preoperative visometric examination. Preoperative variables included Snellen visual acuity, duration of macular detachment, extent of RD, and visometric potential acuity. Reattachment surgery consisted of radial scleral buckling in 33 patients, circumferential scleral buckling and encircling in seven patients, and subretinal fluid drainage in 10 patients. Retinal breaks were treated with cryotherapy or laser photocoagulation. Patients were followed up for at least 6 months after uncomplicated surgery. Best corrected visual acuity measured at any time during follow up was correlated with the preoperative variables. RESULTS: Preoperative visual acuity was less than 20/200 in 37 (93%) of 40 patients. Potential visual acuity of 20/200 or better was measured using the Lotmar visometer in 37 patients (93%). Postoperative visual acuity was correlated significantly with duration of macular detachment (r=0.55; p<0.001), and extent of RD approached statistical significance (r=0.31; p=0.05). There was a higher correlation between postoperative visual acuity and the visometric measurements (r=0.61; p<0.001). CONCLUSIONS: The Lotmar visometer may be a valuable method to estimate visual outcome after uncomplicated scleral buckling surgery in patients with RD involving the macula.     

AIDS／HIV感染合并视网膜脱离

目的探讨获得性免疫缺陷综合征（AIDS）／艾滋病病毒（HIV）感染合并视网膜脱离（RD）的临床特点、药物及手术治疗。方法回顾11例（16眼）AIDS／HIV感染患者RD的实验室检查和临床诊治资料,分析其病因、与细胞免疫的关系、RD发生的高危因素、治疗方法及治疗效果的影响因素。6例（6眼）做了玻璃体视网膜手术。结果13眼孔源性RD由病毒性视网膜炎及可疑病毒性视网膜炎引起,2眼RD由梅毒性葡萄膜炎引起,1眼为原发性孔源性视网膜脱离;发病时,6例CDfT淋巴细胞计数低于100个／μL,3例CD4T淋巴细胞计数高于200个／μL;未手术跟视力无改变,手术眼术后视力有不同程度提高,病程短及单纯性孔源性网脱眼术后矫正视力好于病程长及感染性视网膜炎导致的网脱眼。结论病毒性视网膜炎是CD4^＋T淋巴细胞计数较低的AIDS患者发生孔源性RD的主要病因,原发性孔源性RD及非病毒感染葡萄膜视网膜炎是CD4^＋T淋巴细胞计数较高的AIDS／HIV感染患者RD的主要病因。CD4^＋T淋巴细胞计数较低是RD的高危因素;玻璃体视网膜手术是视网膜解剖复位、改善视力的首选治疗方法。RD的病因、病程及病情严重程度是影响手术后视力的因素。