Lycium barbarum polysaccharides protected human retinal pigment epithelial cells against oxidative stress-induced apoptosis

AIM: To investigate the protective effect and its mechanism of lycium barbarum polysaccharides (LBP) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells. METHODS: ARPE-19 cells, a human retinal pigment epithelial cell lines, were exposed to different concentrations of H2O2 for 24h, then cell viability was measured by Cell Counting Kit-8 (CCK-8) assay to get the properly concentration of H2O2 which can induce half apoptosis of APRE-19. With different concentrations of LBP pretreatment, the ARPE-19 cells were then exposed to appropriate concentration of H2O2, cell apoptosis was detected by flow cytometric analysis. Expression levels of Bcl-2 and Bax were measured by real time quantitative polymerase chain reaction (RT-PCR) technique. RSULTS: LBP significantly reduced the H2O2-induced ARPE-19 cells’ apoptosis. LBP inhibited the H2O2-induced down-regulation of Bcl-2 and up-regulation of Bax. CONCLUSION: LBP could protect ARPE-19 cells from H2O2-induced apoptosis. The Bcl-2 family had relationship with the protective effects of LBP.     

Preconditioning protects the retinal pigment epithelium cells from oxidative stress‐induced cell death

Purpose: The cytotoxic effects of oxidative stress, which play an important role in ocular diseases, are well known. In this study, we investigated the effect of non‐lethal doses of oxidative stress on various cell functions, namely cell viability, cell attachment and cell migration in a widely used retinal pigment epithelium (RPE) cell line (ARPE‐19). Methods: A single exposure to various concentrations of hydrogen peroxide (H₂O₂) was used to establish a dose response for H₂O₂‐induced cell death. Other cellular responses, such as changes in cell attachment and migration, were monitored after exposure to increasing doses. Finally, the effects of preconditioning cells with increasing non‐lethal doses of H₂O₂, with and without a subsequent exposure to lethal doses of H₂O₂, were determined. Results: The optimum dose for inducing cell death in ARPE‐19 cells was between 900 and 1000 μm H₂O₂. Preconditioning the cells with 1, 10 and 50 μm of H₂O₂ provided a dose‐dependent protection against cell death induced by a lethal dose (900–1000 μm ) of H₂O₂. Preconditioning with higher doses caused cells to become more susceptible to the cytotoxic effects of the lethal dose. Although H₂O₂ increased cell attachment in lower doses, it induced a dose‐dependent inhibition of cell attachment to the substrate in higher doses. H₂O₂ did not affect cell migration in sub‐lethal doses. Conclusion: Preconditioning RPE cells with limited exposure to non‐lethal oxidative stress confers significant protection against subsequent H₂O₂‐induced cell death. It also affects cell attachment in a dose‐specific manner. This finding may help in understanding the pathogenesis of diseases in which oxidative stress plays an important role and in determining the suitability of certain treatment strategies, in particular RPE transplantation in the treatment of age‐related macular degeneration.     

视网膜脱离状态下色素上皮细胞的凋亡变异

目的探讨视网膜脱离(RD)后色素上皮(RPE)细胞的凋亡.方法制造有色眼家兔的RD模型,以流式细胞仪(FCM)技术分析RD后28天内RPE细胞的凋亡变化.结果RD后8hRPE细胞凋亡显著增加,第7天达顶峰,凋亡细胞平均数由正常的131.67±35.67上升至1196.2±238,凋亡率由正常的0.70%±0.21%上升至5.98%±1.21%,然后缓慢下降,第28天仍显著高于正常.结论RD可导致RPE细胞的凋亡.RD后28天内,随时间延长,RPE凋亡增加.     

盐酸甲氯芬酯对人视网膜色素上皮细胞增生的影响

目的观察盐酸甲氯芬酯对体外培养的人视网膜色素上皮细胞增生的影响。方法通过MTT比色法和核仁嗜银蛋白染色分析,观察盐酸甲氯芬酯对人视网膜色素上皮细胞增生的影响。结果不同浓度盐酸甲氯芬酯可以促进人视网膜色素上皮细胞增生,在10~1000mg·L-1的范围内呈剂量效应关系,并随时间的延长,促进效应增强。嗜银蛋白染色结果:10mg·L-1盐酸甲氯芬酯作用24h后其胞核内AgNORs的数量增加,平均数为2.0(P=0.029);100mg·L-1盐酸甲氯芬酯作用12h即有胞核内AgNORs的增多,平均数为2.0(P=0.029)。随剂量的增加和时间的延长,胞核内AgNORs的数量增加。结论盐酸甲氯芬酯可以促进人视网膜色素上皮细胞增生,并与剂量和作用时间有一定的关系。     

Cultivation of retinal pigment epithelial cells from human choroidal neovascular membranes in age related macular degeneration

A method is described for cultivating retinal pigment epithelial cells from choroidal neovascular membrane (CNV) specimens that were surgically removed in patients with age-related macular degeneration (AMD). CNV specimens of 43 patients were available for cultivation. They were incubated in supplemented DMEM/Ham's F12 cell culture medium on microporous semipermeable filter membranes. Thirty-four specimens gave rise to cell cultures, 28 of which could be subcultivated for up to 15 passages. The membrane type as classified by fluorescence angiography was compared with cellular growth in vitro. Immunocytochemistry revealed a uniform expression of cytokeratin 18 and vimentin, while factor 8, glial fibrillary acidic protein and alpha smooth muscle actin were absent in all 21 cultures stained. The expression of RPE markers cellular retinaldehyde binding protein (CRALBP) and RPE65 was detected by RT-PCR in all cultures tested. An epithelial character of the cultures was supported by the presence of apical microvilli as determined by electron microscopical studies. Therefore, the cell cultures from CNV in AMD bear characteristics of retinal pigment epithelial cells. For the first time, this cell culture system holds the potential to study human RPE cells in the context of neovascular AMD in vitro.     

Melatonin protects human retinal pigment epithelial (RPE) cells against oxidative stress

Oxidative stress is involved in the pathogenesis of age-related macular degeneration (AMD). Administration of conventional antioxidants has been shown to slow the progression of AMD and vision loss. Melatonin, an endogenous neurohormone produced by the pineal gland and retina, has been reported to be a potent antioxidant and free radical scavenger. In this study we tested whether melatonin can protect retinal pigment epithelial (RPE) cells against hydrogen peroxide (H(2)O(2))-induced cell death. Since mitochondrial DNA (mtDNA) is preferentially susceptible to oxidative damage, we tested whether melatonin can reduce H(2)O(2)-induced mtDNA lesions. A human RPE cell line (ARPE-19) was cultured and exposed to H(2)O(2) (100 and 200 microm) for 1 hr to induce cell death. Prior to H(2)O(2) treatment, cells were treated with various concentrations (0.1-200 microm) of melatonin for 2, 24 or 72 hr. Control cells received either melatonin or ethanol alone. Cell viability, as determined by MTT assay, showed no significant (P>0.05) protection against H(2)O(2) toxicity in cells receiving 2- and 24-hr pretreatment of melatonin at either concentration. However, when melatonin was administered diurnally for 3 consecutive days, this prolonged treatment markedly reduced H(2)O(2)-induced cell death (P>0.05) MtDNA damage, as assessed with quantitative PCR, was significantly decreased (P<0.05) in RPE cells pretreated with melatonin as compared to those without melatonin treatment. These results suggest that melatonin may play a role in protecting RPE cells from oxidative stress.     

ACTA‐EVER lecture 2007 The retinal pigment epithelium: friend or foe?

The retinal pigment epithelium (RPE) plays an important role in the physiology and pathophysiology of the vertebrate retina. The RPE absorbs fluid from the retinal extracellular space, via a proton–lactate–water co‐transport mechanism located in the apical membrane of the epithelium. This mechanism can account for the apparent capability of the RPE to absorb water against an osmotic gradient. RPE cells participate in retinal wound healing. We have created a porcine model of experimental choroidal neovascularization (CNV). In this model, the CNV eventually becomes enveloped by seemingly proliferating RPE cells. By means of 5‐bromo‐2‐deoxyuridine (BrdU) labelling, we studied the proliferation of RPE cells in the porcine eye after experimental posterior pole injury. Surprisingly, we found that only the peripheral RPE cells incorporated the BrdU label, indicating that central injury elicits peripheral RPE proliferation. This might suggest the existence of a peripheral pool of RPE stem cells. RPE cell proliferation plays a role in the pathological wound healing known as proliferative vitreoretinopathy. Antiproliferative agents have been tried to treat this condition but with little success so far. We report on a drug delivery system under development where a prodrug of the antimetabolite 5‐fluoro‐uracil (5‐FU) is suspended in the silicone oil used as a surgical device in the treatment of proliferative vitreoretinopathy (PVR). The theoretical advantage of this approach is that it allows for long contact times between therapeutic, and non‐toxic, concentrations of 5‐FU and the RPE.     

干性ARMD中视网膜色素上皮细胞的作用及损伤机制

年龄相关性黄斑变性(age-related macular degeneration,ARMD)是导致老年人中心视力不可逆丧失的主要原因。ARMD的典型特征是视网膜色素上皮(retinal pigment epithelium,RPE)和脉络膜毛细血管发生退行性改变及黄斑区出现玻璃膜疣。临床上ARMD分为两种亚型：非渗出型(干性或萎缩型)和渗出型(湿性或新生血管型)。该病的发生是年龄、环境、遗传、吸烟、氧化应激和心血管功能障碍等多种因素相互作用的结果。鉴于RPE细胞在ARMD发病中的重要作用,现以RPE细胞为重点,总结了蓝光、吸烟、氧化应激、脂褐素积累、慢性炎症和蛋白质稳态对干性ARMD发病的作用和可能机制,为认识和预防干性ARMD的发生提供新的帮助。     

干性年龄相关性黄斑变性患者视网膜色素上皮细胞损伤机制

年龄相关性黄斑变性(age-related macular degeneration,AMD)是中老年人视力障碍的重要原因之一。AMD分为2型:萎缩型(干性)和渗出型(湿性)。本文从光照损伤、脂褐素积累、氧化损伤、内质网应激4个方面,综述了干性AMD研究中基于视网膜色素上皮细胞损伤的相关研究进展,为进一步探索其发病机制提供依据和方向。     

High glucose‐induced apoptosis in bovine retinal pericytes is associated with transforming growth factor β and βIG‐H3: βIG‐H3 induces apoptosis in retinal pericytes by releasing Arg‐Gly‐Asp peptides

Background: Transforming growth factor β (TGF‐β) plays an important role in diabetic retinopathy. βIG‐H3 is a downstream target molecule of TGF‐β that may participate in the pathogenesis of diabetic retinopathy and in particular in the loss of pericytes during early pathological changes. Methods: We observed bovine retinal pericytes apoptosis and the increased expression of TGF‐β and βIG‐H3 induced by high concentrations of glucose in the cell culture media. An anti‐TGF‐β antibody was used to block glucose‐induced retinal pericytes apoptosis. Retinal pericytes were also transfected with cDNA encodings either wild‐type or mutant βIG‐H3 lacking Arg‐Gly‐Asp (RGD) sequences in order to validate the effects of βIG‐H3 and RGD signalling on retinal pericytes apoptosis. Results: A cell death‐detecting enzyme‐linked immunosorbent assay revealed that 25 mM glucose significantly increased cell death compared with 5.5 mM glucose after 5 or 7 days of exposure (P < 0.01). High glucose significantly increased the TGF‐β levels as compared with 5.5 mM glucose after 5 days, and βIG‐H3 levels after 3, 5 and 7 days of exposure (P < 0.01). TGF‐β increased cell death and βIG‐H3 levels in a dose‐dependent manner, with a maximal effect observed at 1 ng/mL. An anti‐TGF‐β antibody nearly completely blocked high glucose‐induced cell death. Wild‐type βIG‐H3‐transfected cells showed a significant increase in cell death as compared with mutant βIG‐H3‐transfected (Mycb‐c) cells, untransfected or mock‐transfected cells. Conclusion: These results suggest that hyperglycaemia‐induced expression of TGF‐β and βIG‐H3 contributes to accelerated retinal pericytes apoptosis. βIG‐H3 induces pericytes apoptosis through its RGD motif, which may constitute an important pathogenic mechanism leading to pericytes loss in diabetic retinopathy.     

miRNA-146a在视网膜色素上皮细胞老化及老年性黄斑变性中的功能研究

目的 检测miRNA-146a(miR-146a)在视网膜色素上皮细胞(retinal pigment epithelial cells,RPE)衰老及老年性黄斑变性(age-related macular degeneration,AMD)中的表达水平,并探讨其通过抑制VEGF-A表达,参与调控AMD病变进程的机制.方法 qRT-PCR检测年龄为2个月、8个月、12个月、18个月和24个月小鼠的RPE及75岁晚期“湿”性AMD患者眼球中miR-146以及VEGF-A的mRNA表达水平.Western Blotting检测VEGF-A蛋白表达水平.在ARPE-19细胞系中转染miR-146a,通过qRT-PCR及Western Blotting检测miR-146a对VEGF-A mRNA及蛋白水平的影响.结果 在自然衰老的RPE中,miR-146的表达呈现随年龄增加逐渐上升的趋势,与2个月的小鼠相比,在18个月及24个月的小鼠中miR-146a水平上升约8倍及24倍.而VEGF-A的表达呈现下降趋势,与对照相比,24个月的小鼠RPE中的VEGF-A相对水平由2个月时的1.5倍降为0.8倍.但是在AMD患者中,病灶部位的miR-146a较病灶旁侧表达下降14.5倍,VEGF-A表达上升.在ARPE-19细胞中过表达miR-146a抑制了VEGF-A的mRNA及蛋白表达水平.结论 本实验结果将miR-146a与RPE衰老及AMD的发生联系在一起,未来有可能成为RPE衰老及AMD早期诊断的新的分子标记物,为制定临床治疗策略提供参考.     

白藜芦醇对碘酸钠诱导后人视网膜色素上皮细胞凋亡的抑制作用

目的　研究白藜芦醇（resveratrol，RES）对碘酸钠诱导的人视网膜色素上皮（retinal pigment epithelial，RPE）细胞凋亡的抑制作用及机制，探索该药是否可用于干性年龄相关性黄斑变性（age-related macular degeneration,AMD)的治疗。方法　通过碘酸钠诱导的RPE细胞模拟干性AMD。RPE细胞传代培养分为空白对照组、碘酸钠损伤组、RES+碘酸钠损伤组，并观察各组细胞形态。MTT法检测RES对RPE细胞最佳保护作用的浓度以及最佳浓度下对不同浓度碘酸钠损伤的RPE细胞的保护作用。Western blot检测各组细胞的凋亡蛋白Bax和Bcl-2的表达情况。Hosste33342及流式细胞术检测各组细胞的凋亡总量变化以及凋亡种类之间的差别。结果　5 μmol·L^-1 RES对RPE细胞有较强的保护作用且此浓度下对不同损伤程度的RPE细胞都有较强的保护作用。RES+碘酸钠损伤组Bax蛋白的相对表达量（0.45±0.07）明显低于碘酸钠损伤组（0.90±0.09），差异有统计学意义（P<0.05），与空白对照组（0.34±0.06）差异无统计学意义（P>0.05）。RES+碘酸钠损伤组Bcl-2蛋白的相对表达量（0.56±0.14）与碘酸钠损伤组（0.24±0.04）差异有统计学意义（P<0.05），与空白对照组（0.73±0.08）差异无统计学意义（P>0.05）。Hosste33342检测结果显示，RES+碘酸钠损伤组凋亡细胞含量明显低于碘酸钠损伤组（P<0.05），且流式细胞术检测发现，RES+碘酸钠损伤组和碘酸钠损伤组比较，RES+碘酸钠损伤组早期凋亡的细胞含量相对较高，晚期凋亡细胞含量相对较少。结论　RES对碘酸钠诱导损伤的RPE细胞的凋亡具有良好的抑制作用，对干性AMD的治疗具有潜在价值。     

木犀草素对碘酸钠诱导的视网膜色素上皮细胞及视网膜损伤的保护作用

目的　探讨木犀草素对碘酸钠诱导的视网膜色素上皮（retinal pigment epithelial，RPE）细胞及视网膜损伤的保护作用。方法　体外实验选择人RPE细胞（ARPE-19），先将细胞分为对照组、碘酸钠模型组（1 g·L^-1碘酸钠）、木犀草素浓度梯度治疗组（10 μmol·L^-1、25 μmol·L^-1、50 μmol·L^-1、100 μmol·L^-1），MTT法分别检测各组细胞活力。随后将细胞分为对照组，碘酸钠模型组（1 g·L^-1碘酸钠），木犀草素治疗组（50 μmol·L^-1木犀草素），Hoechst33342染色和流式细胞仪检测细胞凋亡情况，Western blotting检测高迁移率族蛋白B1(high mobility group box l，HMGB1)、核因子-κB(nuclear factor kappa B，NF-κB)、cleaved caspase-3相对表达量的变化。体内实验:将30只C57BL/6J雄性小鼠随机分为对照组、碘酸钠模型组和木犀草素治疗组，木犀草素治疗组给予木犀草素预处理1周后，碘酸钠模型组与木犀草素治疗组鼠尾静脉给予碘酸钠造模，木犀草素治疗组继续如前给药4周，光学相干断层扫描 (optical coherence tomography，OCT)测量视网膜厚度，HE染色观察形态学变化并统计玻璃膜疣数量。结果　体外实验:MTT显示，碘酸钠模型组吸光度(A)值低于对照组（P<0.05），木犀草素浓度梯度治疗组A值均高于碘酸钠模型组（P<0.05）。Hoechst33342染色显示，碘酸钠模型组细胞凋亡率高于对照组（P<0.05），木犀草素治疗组细胞凋亡率低于碘酸钠模型组（P<0.05）。流式细胞计数显示，碘酸钠模型组细胞凋亡率高于对照组（P<0.05），木犀草素治疗组细胞凋亡率低于碘酸钠模型组（P<0.05）。Western blotting检测显示，碘酸钠模型组HMGB1、NF-κB、cleaved caspase-3相对表达量均高于对照组（均为P<0.05），木犀草素治疗组HMGB1、NF-κB、cleaved caspase-3相对表达量均低于碘酸钠模型组（均为P<0.05）。体内实验:OCT显示，对照组各层结构排列均一整齐，与碘酸钠模型组相比，木犀草素治疗组RPE层高反射点减少，光感受器和外核层排列紊乱程度减轻，视网膜相对厚度增加（P<0.05）。HE染色显示，对照组各层细胞排列整齐，密度均匀；与碘酸钠模型组相比，木犀草素治疗组外核层褶皱程度减轻，RPE层连续性增加，棕黑色颗粒（玻璃膜疣）沉积数量减少（P<0.05）。结论　木犀草素通过降低HMGB1表达减轻NF-κB介导的炎症抑制碘酸钠诱导的RPE细胞凋亡并保护视网膜免受损伤。     

内质网应激在氧化低密度脂蛋白诱导的人RPE细胞凋亡中的作用

目的：观察内质网应激(ERS)在氧化低密度脂蛋白(OxLDL)诱导的人视网膜色素上皮(RPE)细胞凋亡中的作用。

方法：人RPE细胞系ARPE19采用低糖DMEM培养基和10%胎牛血清进行常规培养。实验分为3组：对照组(常规培养的ARPE19)、OxLDL组(加入5、10、25、50、100μg/mL OxLDL)和LDL组(加入5、10、25、50、100μg/mL LDL)培养24h。分别采用细胞计数试剂盒(CCK8)检测各组细胞活性,流式细胞仪检测凋亡比例,蛋白质印迹(Western blotting)检测ERS相关蛋白及凋亡相关酶的表达。激光共聚焦显微镜观察人RPE细胞吞噬红色荧光探针Dil标记的氧化低密度脂蛋白(Dil-OxLDL)情况。

结果：CCK8结果显示：对照组细胞存活率为(100±5.637)%,加入5、10、25、50、100μg/mL OxLDL后细胞活力分别为(105.298±9.395)%、(97.106±5.417)%、(77.015±4.055)%、(67.613±3.853)%和(43.872±9.532)%(P<0.05); 加入5、10、25、50、100μg/mL LDL后细胞活力分别为(97.55±6.217)%、(99.640±3.586)%、(90.495±2.786)%、(83.552±9.171)%和(90.910±1.429)%(P>0.05)。流式细胞仪结果显示浓度为25μg/mL的OxLDL会明显诱导细胞凋亡,对照组、OxLDL组(25μg/mL)和LDL(25μg/mL)组凋亡率分别是(5.271±0.519)%、(41.23±1.686)%和(13.07±2.579)%(P<0.01); Western blotting结果显示OxLDL(25μg/mL)组的ERS相关蛋白和凋亡相关酶的表达量明显高于对照组与LDL组(Caspase-12：F=50.53, P<0.05; GRP78：F=55.60, P<0.05; CHOP：F=38.22, P<0.05; XBP-1：F=53.94, P<0.05; ATF6：F=20.01, P<0.05),而LDL组(25μg/mL)和对照组之间无差异(P>0.05)。

结论：ERS参与了OxLDL诱导的人RPE细胞凋亡,调控ERS可能抑制人RPE细胞凋亡,从而治疗RPE细胞凋亡相关疾病。     

Subretinal surgery and transplantation of autologous pigment epithelial cells in retinal angiomatous proliferation

Purpose: The aim of this study was to examine whether the presence of retinal angiomatous proliferation (RAP) is a prognostic factor in subretinal surgery with transplantation of a suspension of autologous retinal pigment epithelial (RPE) cells. Methods: Eyes that had been followed for at least 12 months after subretinal surgery were reviewed retrospectively and assigned to group 1 (presence of RAP) or group 2 (lesions without RAP). Main outcome measures were logMAR distance acuity and lesion size at 12 months. Results: A total of 68 eyes of 68 patients were included; 28 were assigned to group 1 and 40 to group 2. A total of 43% of patients were male and 57% were female. Their mean age was 77.8 years. Mean distance acuity was 1.02 logMAR at baseline and 1.06 logMAR at month 12. Mean lesion size was 27.9 mm² at baseline and 29.9 mm² at month 12. The differences between the groups were not significant. Conclusions: The presence of RAP did not significantly influence the outcome after subretinal surgery with transplantation of autologous RPE cells. Other than age, preoperative lesion size and distance acuity were the only predictive factors for postoperative results.     

Enhanced expression of glutathione-S-transferase A1-1 protects against oxidative stress in human retinal pigment epithelial cells

Glutathione-S-transferases (GSTs) play an important role in protection mechanisms against oxidative stress. We sought to determine whether over-expression of human GSTA1-1 in RPE cells is able to attenuate H(2)O(2)-induced oxidative stress. SV40-transformed human fetal RPE cells were stably transfected with pRC/hGSTA1-1 vector which carries a full-length of human GSTA1-1 cDNA. The control RPE cells were either non-transfected or transfected with control vector pRC. Expression of hGSTA1-1 protein in these cells was confirmed by Western blot and immunocytochemical analyses. The protective effects of hGSTA1-1 on cell viability and mitochondrial DNA (mtDNA) damage caused by H(2)O(2) were examined with MTT assay and quantitative PCR (QPCR), respectively. The hGSTA1-1 transfected RPE cells exhibited a similar morphology and growth rate as control RPE cells. Immunocytochemical analysis showed robust expression hGSTA1-1 in hGSTA1-1 transfected cells versus background staining in control cells. Western blotting of protein extracts from cells transfected with hGSTA1-1 revealed a 26 kDa protein band which corresponds to the size of recombinant mature hGSTA1-1. The active GST present in the hGSTA1-1 transfected cells was approximately three times higher than in control cells. The MTT assay showed a significantly greater viability of hGSTA1-1 cells in response to H(2)O(2) (100 and 200 microm) compared to control cells (p<0.05). QPCR indicated that mtDNA damage was significantly decreased in hGSTA1-1 cells than in control cells (p<0.05). Human GSTA1-1 transfection protect against RPE cell death and mtDNA damage caused by H(2)O(2), suggesting an important role of GST in protection against oxidative stress in RPE cells.     

贝伐单抗对视网膜色素上皮细胞抗氧化功能的影响

目的 观察贝伐单抗对人视网膜色素上皮(retinal pigment epithelium,RPE)细胞抗氧化功能的影响,以探讨抗新生血管内皮生长因子(vascular endothelial growth factor,VEGF)制剂治疗年龄相关性黄斑变性后黄斑部萎缩的可能机制.方法 用含终浓度0.25g·L-1贝伐单抗的DMED/F12培养液培养人RPE细胞系ARPE-19细胞,根据处理时间的不同分为Oh组(对照组)、12 h组、24 h组、48 h组、72 h组共5组,加入H2O2诱导氧化应激反应.用CCK-8法检测细胞活性,MitoSox Red荧光染色检测细胞内线粒体活性氧(reactive oxygen species,ROS)产生水平,JC-1荧光染色检测细胞线粒体膜电位的变化;分别用逆转录聚合酶链反应(RT-PCR)及免疫蛋白印迹法(Western blot)检测各组促氧化因子NADPH氧化酶4(NADPH oxidase 4,NOX4)和抗氧化因子血红素氧合酶-1(heme oxygenase 1,HO-1) mRNA和蛋白的表达水平.结果 CCK-8检测结果显示:上述处理对细胞活性无显著影响,Oh组、12 h组、24h组、48h组和72 h组细胞活性分别为(100.2±3.3)％、(99.2±2.7)％、(102.5±6.4)％、(103.9±3.7)％和(103.6±3.3)％,差异无统计学意义(P＞0.05);与对照组相比,12 h、24h、48 h、72 h组细胞内ROS水平上升,差异有统计学意义(P＜0.05);线粒体膜电位在12 h、24h、48 h、72 h组均较对照组降低,差异有统计学意义(P＜0.01),48 h达最低,72 h时显著提高,但仍低于对照组.RT-PCR和Western blot检测结果显示:与对照组比较,NOX4 mRNA和蛋白的表达在12 h、24h、48 h和72 h组均上升,而且在24h表达最高,之后明显下降,但仍高于对照组,差异有统计学意义(P＜0.01).与对照组比较,HO-1 mRNA的表达在24h、48 h和72 h组均下降,而HO-1蛋白的表达在48 h和72 h组下降,差异有统计学意义(P＜0.05).结论 临床浓度的贝伐单抗可以降低RPE的抗氧化功能,可能是长期抗VEGF治疗后黄斑部进行性萎缩的原因之一.