Indocyanine green induces apoptosis in human retinal pigment epithelial cells

PURPOSE: To examine whether indocyanine green (ICG) dye induces apoptosis in human retinal pigment epithelial (RPE) cells. DESIGN: Laboratory investigation. METHODS: Pure cultures of human RPE cells were isolated. Retinal pigment epithelial cells were incubated with different concentrations of ICG dye (1 mg/ml, 5 mg/ml, or 20 mg/ml) for 30 minutes. The rate of RPE cell apoptosis was assessed with Annexin V-FITC staining and propidium iodide (PI) by flow cytometry. RESULTS: Retinal pigment epithelial cells maintained their monolayer morphology after incubation with ICG dye. However, ICG induced a statistically significant amount of apoptosis in RPE cells at all the concentrations (1 mg/ml, 5 mg/ml, and 20 mg/ml) after 30 minutes of incubation (P <.05). The solvent solution alone (without the ICG dye) did not induce any significant apoptosis in RPE cells, when compared with culture medium. CONCLUSIONS: The incubation of RPE cells with ICG dye increased the number of apoptotic RPE cells in vitro. Our findings indicate that the decision over the intravitreal application of ICG dye needs to be made with caution.     

Trypan blue induces apoptosis in human retinal pigment epithelial cells

PURPOSE: To examine whether trypan blue dye induces apoptosis in human retinal pigment epithelium cells. DESIGN: Laboratory investigation. METHODS: Pure cultures of human retinal pigment epithelium cells were isolated. The cells were incubated with different concentrations of trypan blue (0.5%, 0.10%, and 0.05%) for either 5 or 30 minutes. The rate of retinal pigment epithelium cell apoptosis was assessed with Annexin V-PE staining and flow cytometry. RESULTS: Trypan blue induced a statistically significant amount of apoptosis in retinal pigment epithelium cells at all the concentrations (0.5%, 0.10%, and 0.05%) (P <.05). The increase in incubation time (from 5 to 30 minutes) led to an increase in the number of apoptotic retinal pigment epithelium cells. CONCLUSION: The incubation of retinal pigment epithelium cells with trypan blue increased the number of apoptotic retinal pigment epithelium cells in vitro. Our results suggest that decisions regarding the intravitreal application of trypan blue dye need to be made with caution.     

缺氧诱导体外培养人视网膜色素上皮细胞凋亡鲻

目的:了解缺氧条件下体外培养人视网膜色素上皮 (retinal pigment epithelium,RPE)细胞的凋亡情况.方法:将培养的人RPE细胞置于含10mL/L O2、50mL/L CO2和940mL/L N2的培养箱内建立缺氧模型.于缺氧后1,3,6,12,24h,利用扫描电镜、透射电镜、脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)技术和流式细胞仪测定RPE细胞凋亡水平.结果:常氧状态下RPE细胞生长良好,几乎没有凋亡(TUNEL法测凋亡指数为1.2).缺氧条件下,RPE细胞发生了不同程度的凋亡,缺氧后3h凋亡水平达峰值(凋亡指数为34.43).结论:缺氧可导致体外培养的人RPE细胞凋亡,提示RPE凋亡可能参与了某些缺血缺氧性眼病的发生.     

缺氧诱导体外培养人视网膜色素上皮细胞凋亡

目的:了解缺氧条件下体外培养人视网膜色素上皮(retinal pigment epithelium,RPE)细胞的凋亡情况。方法:将培养的人RPE细胞置于含10mL/LO2、50mL/LCO2和940mL/LN2的培养箱内建立缺氧模型。于缺氧后1,3,6,12,24h,利用扫描电镜、透射电镜、脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucle-otidyl transferase mediated nick end labeling,TUNEL)技术和流式细胞仪测定RPE细胞凋亡水平。结果:常氧状态下RPE细胞生长良好,几乎没有凋亡(TUNEL法测凋亡指数为1.2)。缺氧条件下,RPE细胞发生了不同程度的凋亡,缺氧后3h凋亡水平达峰值(凋亡指数为34.43)。结论:缺氧可导致体外培养的人RPE细胞凋亡,提示RPE凋亡可能参与了某些缺血缺氧性眼病的发生。     

Oxidant-induced apoptosis in cultured human retinal pigment epithelial cells

PURPOSE: To determine the mechanism of oxidant-induced cell death in cultured human retinal pigment epithelium (hRPE). METHODS: Cultured hRPE cells were treated with different concentrations of a chemical oxidant, t-butylhydroperoxide (tBH), for different periods of time. Apoptosis was determined with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and flow cytometry. Mitochondrial membrane potential (mtdelta psi) was measured by rhodamine 123 staining and subsequent flow cytometry. Release of mitochondrial cytochrome c (cyt c) and cleavage of procaspase 3 and caspase substrates were determined by western blot analysis. RESULTS: t-Butylhydroperoxide caused time- and dose-dependent activation of apoptosis in hRPE, indicated by characteristic morphologic changes; TUNEL-positive labeling; phosphatidylserine (PS) exposure; and procaspase 3, poly(ADP-ribose)polymerase, lamin, and tubulin cleavage. An early decrease of mtdelta psi was observed before caspase activation, together with the release of mitochondrial cyt c. CONCLUSIONS: Results indicate that tBH can induce apoptosis in hRPE, probably by triggering the mitochondrial permeability transition, which results in swelling and release of mitochondrial intermembrane proteins.     

全反式视黄酸诱导人视网膜色素上皮细胞凋亡的实验研究

目的探讨梯度浓度全反式视黄酸(all-trans retinoic acid,ATRA)诱导人视网膜色素上皮细胞凋亡的作用。方法采用流式细胞术检测视网膜色素上皮细胞凋亡、活性氧(ROS)活性。采用实时定量聚合酶链反应和Western blot,从蛋白水平检测梯度浓度ATRA对ARPE-19细胞内质网应激(endoplasmic reticulum stress,ERS)标记蛋白表达的影响。观察抗氧化剂NAC及ERS抑制剂Salubrinal抑制ARPE-19细胞诱导ROS和ERS的作用。结果 CCK-8检测结果显示:ATRA处理ARPE-19细胞24 h和48 h后的半抑制浓度(IC₅₀)分别为13.88μmol·L^-1和11.99μmol·L^-1。流式细胞术检测结果显示,10.0μmol·L^-1、15.0μmol·L^-1、20.0μmol·L^-1ATRA处理后细胞凋亡水平均较对照组显著上升,差异均有统计学意义(均为P<0.001);2.5μmol·...     

Regulation of thioredoxin by ceramide in retinal pigment epithelial cells

The purpose of this study was to determine the expression, regulation and signaling of a key redoxin family member thioredoxin 1 (Trx1) in normal, oxidant-stimulated and growth factor-pretreated RPE cells. Trx1 is expressed in early passage, human RPE cell cultures. RPE cells exposed to C₂-ceramide for 24 h showed no significant change in expression of Trx1 vs. controls with and without pretreatment for 24 h with hepatocyte growth factor (HGF). Neither hypoxia from 1% O₂ or from CoCl₂ exposure resulted in any alteration in Trx1 expression in RPE cells. C₂-ceramide treatment caused translocation of Trx1 from cytosol to the nucleus, which was abolished by pre-treatment of cells with a p38 MAPK-specific inhibitor. Furthermore, the gene and protein expression of thioredoxin interacting protein (Txnip) increased with ceramide treatment and was significantly (p < 0.001) elevated with HGF preincubation vs. untreated controls. Prominent protection from ceramide-induced RPE cell death by exogenous rTrx1 was demonstrated. Although Trx1 directly interacts with its inhibitor, Txnip, p38 inhibition does not appear to have a role in this interaction. We found no direct interaction between apoptosis signal regulating kinase (ASK-1) and Txnip under the same experimental conditions. In summary, our data demonstrate the expression of Trx1 and Txnip in human RPE cells. Ceramide treatment results in translocation of Trx1 to the nucleus, and upregulation of Txnip expression; exogenous rTrx1 protects from ceramide-induced cell death. These results suggest that Trx1 and Txnip play an important role in the response of RPE to ceramide toxicity.     

蓝光诱导体外培养的人视网膜色素上皮细胞凋亡

目的 观察蓝光光照对体外培养的人视网膜色素上皮（RPE）细胞凋亡的影响。 方法 用蓝光照射体外培养的人RPE细胞，分为3组，A组：不同光照强度；B组：同一光照强度，不同光照时间；C组：同一光照强度和光照时间，不同细胞培养终止时间。利用末端脱氧核酸转移酶介导的原位缺口末端标记（TUNEL）、荧光素标记的连接素V/碘化吡啶（Annexin V-FITC/PI）双染流式细胞检测、透射电子显微镜等手段观察细胞凋亡。 结果 TUNEL染色阳性细胞的体积缩小变圆，细胞核浓缩，边聚成新月形或帽状，细胞核碎裂成数个，细胞膜出胞。透射电子显微镜观察发现细胞内线粒体肿胀，线粒体内膜嵴消失；粗面内质网扩张；溶酶体增加，内含代谢产物。A组低于一定阈值［（500±100）lx］的蓝光光照对RPE细胞损伤较轻，细胞凋亡及坏死随光照强度的增强而增加；B组随着光照时间延长，RPE细胞凋亡数量没有增加，而细胞坏死逐渐增加；C组光照后6、12h，损伤以细胞凋亡为主，但随着光照时间延长，凋亡继发坏死细胞和坏死细胞明显增加。 结论 蓝光照射可引起体外培养的人RPE细胞损伤，损伤形式有凋亡、凋亡继发坏死及坏死；损伤程度呈光照强度和光照时间依赖性。 (中华眼底病杂志, 2005, 21: 384-387)     

Mechanisms of apoptosis in retinal pigment epithelial cells

目的了解诱导视网膜色素上皮（ＲＰＥ）细胞在体外发生凋亡的因素，以推测该细胞凋亡在体内发生的可能机理。方法培养的人ＲＰＥ细胞分别给予低氧（３％），肿瘤坏死因子（ＴＮＦ），蛋白激酶Ｃ（ＰＫＣ）抑制剂及抗Ｆａｓ抗体处理２４～７２ｈ，同时也观察了纤维母细胞生长因子（ｂＦＧＦ）对体外凋亡诱导因子所致ＲＰＥ细胞凋亡的保护作用。以琼脂糖ＤＮＡ电泳及流式细胞计数法检测细胞凋亡，采用免疫组化染色及流式细胞计数法决定Ｆａｓ在ＲＰＥ细胞的表达。结果所用的细胞凋亡诱导剂均可使ＲＰＥ细胞发生典型的凋亡（ＤＮＡ片段形成），ｂＦＧＦ具有较强的抑制ＲＰＥ细胞凋亡发生的作用。３Ｈ胸腺嘧啶摄入表明，所有细胞凋亡诱导剂均能抑制ＲＰＥ细胞的生长。结论不同的诱导因子可诱导ＲＰＥ细胞在体外发生凋亡，其发生与多种凋亡信号系统调控和交互反应有关，因此，调节ＲＰＥ细胞凋亡可能会给某些视网膜变性类疾病提供新的治疗手段。     

Hydrogen peroxide stimulates apoptosis in cultured human retinal pigment epithelial cells.

PURPOSE: To determine whether hydrogen peroxide (H2O2), a physiological mediator of oxidative stress induces apoptosis in retinal pigment epithelial (RPE) cells. METHODS: To demonstrate that oxidatively stressed retinal pigment epithelial cells undergo apoptosis consequential to mitochondrial dysfunction, biochemical parameters of apoptosis were determined in cultured cells after treatment with 50-200 mM H2O2 for different times. Caspase-3 protease activity was determined from hydrolysis of DEVD-rho-nitroanilide. Expression of the anti-apoptotic protein, bcl-2 and the pro-apoptotic proteins p53 and p21 were analyzed by western blotting. RESULTS: Caspase-3 activity significantly increased in cells exposed to H2O2. Also, the expression of bcl-2 in cells treated with 200 microM H2O2 was diminished, whereas expression of p53 and p21waf-1 was increased compared to the controls. CONCLUSIONS: Exposure of retinal pigment epithelial cells to concentrations of H2O2 that cause in vitro mitochondrial DNA damage also promotes apoptosis.     

N-甲基-D-门冬氨酸诱导体外培养的视网膜色素上皮细胞凋亡

目的　探讨用N-甲基-D-门冬氨酸(N-methyl-D-aspartate,NMDA)诱导体外目的　探讨用 N-甲基 -D-门冬氨酸 (N-methyl-D-aspartate,NMDA)诱导体外培养的人胚胎视网膜色素上皮 (retinal pigment epithelium,RPE)细胞的凋亡。方法　加入终浓度分别为 1 0、50、1 0 0、2 0 0、3 0 0 μmol· L-1的 NMDA作用 72 h;3 0 0 μmol· L-1NM-DA作用 1 2、2 4、3 6、48、72 h后用透射电镜定性观察凋亡细胞 ,并用吖啶橙荧光染色进行定量和定性分析。结果　经 2 0 0、3 0 0 μmol· L-1NMDA作用 72 h后 RPE细胞凋亡指数分别为 (51 .0 0 0± 1 .4491 ) %和 (74.0 0 0± 1 .71 2 7) %与对照组 (2 .0 0 0 0± 0 .42 1 6) %相比 ,差异有显著性 (t=1 0 .0 66、1 4.790 ,P=0 .0 0 0、0 .0 0 0 ) ,随着 NMDA浓度的增加 ,RPE细胞凋亡数逐渐增多 ;3 0 0 μmol·L-1NMDA作用 2 4、3 6、48、72 h后 RPE细胞凋亡指数分别为(9.0 0 0 0± 0 .73 79) %、 (1 3 .0 0 0 0± 1 .0 593 ) %、(2 5.0 0 0 0± 0 .70 71 ) %、(53 .0 0 0 0±0 .82 3 3 ) %与对照组 (2 .0 0 0 0± 0 .42 1 6) %相比差异有显著性 (t=2 .0 1 4、3 .1 65、6.61 8、1 4.676,P=0 .0 50、0 .0 0 3、0 .0 0 0、0 .0 0 0 )。结论　 NMDA能诱导体外培养的人胚胎 RPE细胞     

Adrenomedullin in cultured human retinal pigment epithelial cells

PURPOSE: To determine whether adrenomedullin (ADM), a vasorelaxant peptide is produced and secreted by human retinal pigment epithelial (RPE) cells, whether ADM expression is regulated by inflammatory cytokines and a growth factor, and whether ADM has proliferative effects on these cells. METHODS: Production and secretion of ADM by cultured human RPE cells were examined by Northern blot analysis and radioimmunoassay. Regulation of the ADM expression by basic fibroblast growth factor, interferon (IFN)-gamma, tumor necrosis factor-alpha, interleukin (IL)1beta, or all-trans-retinoic acid was studied. In addition, proliferative effects of ADM on human RPE cells were examined by modified 3-(4,5-dimetylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: ADM mRNA was expressed constitutively in all three human RPE cell lines (F-0202, D407, and ARPE-19) examined. Immunoreactive ADM was detected in the cultured media by radioimmunoassay. Sephadex G-50 column chromatography of the cultured medium showed a single peak eluting in the position of ADM-(1-52). Treatment with IFN-gamma or IL-beta increased ADM mRNA levels and immunoreactive-ADM levels in the medium in dose- and time-dependent manners in ARPE-19 cells. Exogenously added ADM increased the number of F-0202 cells and ARPE-19 cells, and the treatment with ADM antibody or ADM-(22-52) (an ADM antagonist) decreased it. CONCLUSIONS: Human RPE cells produced and secreted ADM. IFN-gamma and IL-1beta induced ADM expression in ARPE-19 cells. Furthermore, ADM stimulated proliferation of RPE cells. These results raise the possibility that ADM is related to the pathophysiology of some inflammatory and proliferative ocular diseases.     

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.     

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.