Transglutaminase participates in UVB-induced cell death pathways in human corneal epithelial cells

PURPOSE: Ultraviolet light (UVB) is known to cause apoptosis in human corneal epithelial cells. This study evaluates the role of transglutaminase in regulating tumor necrosis factor (TNF) receptor clustering as well as caspase activation in UVB-induced apoptosis in human corneal epithelial cells. METHODS: A human corneal epithelial cell line was used. A single dose of UVB (20 mJ/cm2) was used as a stimulus. Cell viability and cell death were investigated by MTT, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL), and caspase-3 assays. Immunofluorescent staining was used to investigate TNF receptor-I clustering at various time intervals after UVB. Short interfering RNA was used to knock down transglutaminase-2 expression. Fluorescein-cadaverine uptake was used to assess transglutaminase activity. A noncovalent peptide delivery system was used to transfect guinea pig liver transglutaminase into corneal epithelial cells. RESULTS: UVB increased transglutaminase activity, reduced cell viability, and increased TUNEL staining. UVB or TNF-alpha promoted TNF-receptor-I clustering, a process inhibited by the transglutaminase inhibitor, mono-dansyl cadaverine. UVB also increased activated caspase-3, in a manner suppressible by mono-dansyl cadaverine. Intracellular delivery of exogenous transglutaminase markedly increase caspase-3 activation compared with the vehicle control. CONCLUSIONS: Transglutaminase enzymatic activity is involved in corneal epithelial cell death after UVB and appears to participate in two steps regulating this process, clustering of TNF receptor-I and caspase-3 activation.     

紫外线诱导小鼠角膜核因子-κB及肿瘤坏死因子-α的表达

目的:探讨紫外线B光(UVB)照射小鼠角膜后核转录因子(NF-κB)、肿瘤坏死因子-α(TNF-α)的表达变化及NF-κB在角膜损伤中的意义。方法:ICR小鼠随机分为对照组、低剂量UVB照射组(300mJ/cm2)及高剂量UVB照射组(1200mJ/cm2),裂隙灯下观察角膜病变,评分以判断角膜损伤程度。UVB照射后不同时间点(6h、24h及72h)分别取角膜,凝胶电泳迁移法(EMSA)检测角膜NF-κB的活性,酶联免疫吸附(ELISA)测定角膜TNF-α的表达水平,光镜及电镜检查角膜的病理改变。结果:低剂量照射组角膜基质轻度水肿,在72h内基本消退,高剂量照射组角膜基质混浊明显增强且持久。正常对照组小鼠角膜NF-κB的活性水平低,照射组角膜组织出现NF-κB表达的活化,并随剂量的增加活性明显增加,不同剂量组间差异有显著统计学意义。同时,照射后角膜组织TNF-α的表达也明显增强,其变化趋势与NF-κB的活性变化类似。电镜显示低剂量组仅角膜上皮及浅层基质细胞受损,高剂量组角膜损伤累积全基质细胞及内皮细胞。结论:UVB照射小鼠角膜后激活NF-κB,并引发促炎性细胞因子TNF-α的表达。随着角膜损伤程度的加重,NF-κB的活性水平增强,提示NF-κB的激活可能在紫外线造成的角膜损伤中起着重要的作用     

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.     

Stratified corneal limbal epithelial cells are protected from UVB-induced apoptosis by elevated extracellular K⁺

The goal of this study was to determine whether elevated [K⁺] protects stratified corneal epithelial cells from entering apoptosis following exposure to ambient levels of UVB radiation. Human corneal limbal epithelial (HCLE) cells were stratified to form multilayered constructs in culture. The cells were exposed to UVB doses of 100–250 mJ/cm² followed by incubation in medium with 5.5–100 mM K⁺. The protective effect of K⁺ was determined by measuring the caspase-3 and -8 activity and TUNEL staining of the stratified HCLE constructs. In response to UVB exposure, activation of apoptotic pathways peaked at 24 h. Caspase-8 in stratified cells was activated by exposure to UVB at 100–250 mJ/cm², and activity was significantly reduced in response to 50 or 100 mM K⁺. Caspase-3 was activated in the stratified cells in response to 100–250 mJ/cm² UVB and showed a significant reduction in activity in response to 25, 50 or 100 mM K⁺. DNA fragmentation, as indicated by TUNEL staining, was elevated after exposure to 200 mJ/cm² UVB, and decreased following incubation with 25–100 mM K⁺. These results show that in a culture system that models the intact corneal epithelium, elevated extracellular K⁺ can reduce UVB-induced apoptosis which is believed to be initiated by loss of K⁺ from cells. This is the basis of damage to the corneal epithelium caused by UVB exposure. Based on these observations it is suggested that the relatively high K⁺ concentration in tears (20–25 mM) may play a role in protecting the corneal epithelium from ambient UVB radiation.     

Elevated extracellular K inhibits apoptosis of corneal epithelial cells exposed to UV-B radiation

The goal of this study was to determine if the high [K⁺] in tears, 20-25 mM, serves to protect corneal epithelial cells from going into apoptosis after exposure to ambient UV-B radiation. Human corneal-limbal epithelial (HCLE) cells in culture were exposed to UV-B at doses of 50-200 mJ/cm² followed by measurement of K⁺ channel activation and activity of apoptotic pathways. Patch-clamp recording showed activation of K⁺ channels after UV-B exposure at 80 mJ/cm² or 150 mJ/cm² and a decrease in UV-induced K⁺ efflux with increasing [K⁺]_o. The UV-activated current was partially blocked by the specific K⁺ channel blocker, BDS-1. DNA fragmentation, as measured by the TUNEL assay, was induced after exposure to UV-B at 100-200 mJ/cm². DNA fragmentation was significantly decreased when cells were incubated in 25, 50 or 100 mM K_o⁺ after exposure to UV-B. The effector caspase, caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm², but there was a significant decrease in activation when the cells were incubated in 25, 50 or 100 mM K_o⁺ following exposure to UV-B. A decrease in mitochondrial potential, a possible activator of caspase-3, occurred after exposure to UV-B at 100-200 mJ/cm². This decrease in mitochondrial potential was prevented by 100 mM K_o⁺; however, 25 or 50 mM K_o⁺ provided minimal protection. Caspase-9, which is in the pathway from mitochondrial potential change to caspase-3 activation, showed little activation by UV-B radiation. Caspase-8, an initiator caspase that activates caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm², and this UV-activation was significantly reduced by 25-100 mM K_o⁺. The data show that the physiologically relevant [K⁺]_o of 25 mM can inhibit UV-B induced activation of apoptotic pathways. This suggests that the relatively high [K⁺] in tears reduces loss of K⁺ from corneal epithelial cells in response to UV exposure, thereby contributing to the protection of the ocular surface from ambient UV radiation.     

Platelet-activating factor (PAF) enhances apoptosis induced by ultraviolet radiation in corneal epithelial cells through cytochrome c-caspase activation

PURPOSE: To examine the role of platelet-activating factor (PAF) on apoptosis of corneal epithelial cells exposed to radiation. METHODS: Rabbit corneal epithelial (RCE) and human corneal epithelial (HCE) cells were exposed to UVC radiation and then to carbamyl PAF (cPAF) for different increments of time. The PAF antagonist BN50739 was added 30 min before cPAF. The caspase inhibitors Ac-DEVD-CHO and Ac-YVAD-CHO were added 1 h before, and the phospholipase A(2) (PLA(2)) inhibitor MAFP was added 3 h before UVC irradiation. FITC-dUTP TUNEL and DAPI staining were performed to assess the percentage of apoptotic cells. DNA ladder analysis was used to investigate apoptosis induced by different intensities of UVC (50-600 J/m(2)) with or without cPAF. Caspase activation and release of cytochrome c from mitochondria to cytosol were determined by Western blot. RESULTS: While only 2.7% of RCE cells were DAPI positive in controls incubated for 12 h, 44% of cells were stained positive 4 h after irradiation; these values increased to 63% in the presence of cPAF. Cells incubated with cPAF alone were similar to controls. TUNEL staining and DNA laddering showed also increased in apoptosis after PAF treatment of UV-irradiated cells and BN50739 blocked the effect of cPAF. cPAF increased caspase-3 activation induced by UV irradiation in HCE cells. Cytochrome c release from mitochondria to cytosol was observed 30 min after irradiation. cPAF almost doubled the release of cytochrome c at 30 min and 1 h. Here, too, BN50739 blocked the PAF effect. No release of cytochrome c by PAF was seen in non-irradiated cells, even at higher concentrations. MAFP caused a decrease in cytochrome c release from UV-treated cells, and caused an even greater inhibition of cytochrome c release in cells stimulated with PAF. CONCLUSIONS: PAF increases RCE and HCE apoptosis caused by UV irradiation by stimulating PLA(2), producing an early release of cytocrome c from mitochondria and activating caspase-3 by a receptor-mediated mechanism. This accelerating effect of PAF on the apoptotic cascade only occurred when corneal epithelial cells had been previously damaged by UV radiation.     

Thymosin-beta4 inhibits corneal epithelial cell apoptosis after ethanol exposure in vitro

PURPOSE: The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta(4)) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of Tbeta(4) in preventing mitochondrial disruption and in inhibiting caspase-mediated apoptosis was examined. METHODS: Nontransformed human corneal epithelial cells (HCECs) at passage 4 were untreated or treated with ethanol (20% for 20 seconds) or a combination of ethanol and Tbeta(4). The cells were allowed to recover from ethanol treatment for 24 hours. Mitochondrial membrane integrity and the release of cytochrome c to the cytoplasm were assessed using microscopy, Western blot, and ELISA. Bcl-2 expression and cell proliferation were measured using ELISA. Colorimetric activity assays were completed for caspase-2, -3, -8, and -9. RESULTS: Tbeta(4) treatment decreased deleterious mitochondrial alterations, significantly decreased cytochrome c release from mitochondria, and increased Bcl-2 expression in ethanol-exposed human corneal epithelial cells. In ethanol-exposed corneal epithelium Tbeta(4) treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. Tbeta(4) treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure. CONCLUSIONS: Tbeta(4) plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. Tbeta(4) may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases.     

Cytotoxic effect and possible mechanisms of Tetracaine on human corneal epithelial cells in vitro

AIM: To demonstrate the cytotoxic effect and possible mechanisms of Tetracaine on human corneal epithelial (HCEP) cells in vitro. METHODS: In vitro cultured HCEP cell were treated with Tetracaine hydrochloride at different doses for different times, and their morphology, viability, and plasma membrane permeability were detected by light microscopy, methyl thiazolyl tetrazolium (MTT) assay, and acridine orange (AO)/ethidium bromide (EB) staining, respectively. Their cell cycle progression, phosphatidylserine orientation in plasma membrane, and mitochondrial membrane potential (MTP) were assessed by flow cytometry. DNA fragmentation, ultrastructure, caspase activation, and the cytoplasmic apoptosis inducing factor (AIF) and cytochrome c (Cyt. c) along with the expression of B-cell lymphoma-2 (Bcl-2) family proteins were examined by gel electrophoresis, transmission electron microscope, enzyme linked immunosorbent assay (ELISA), and Western blot, respectively. RESULTS: After exposed to Tetracaine at doses from 10.0 to 0.3125 g/L, the HCEP cells showed dose- and time-dependent morphological abnormality and typical cytopathic effect, viability decline, and plasma membrane permeability elevation. Tetracaine induced phosphatidylserine externalization, DNA fragmentation, G1 phase arrest, and ultrastructural abnormality and apoptotic body formation. Furthermore, Tetracaine at a dose of 0.3125 g/L also induced caspase-3, -9 and -8 activation, MTP disruption, up-regulation of the cytoplasmic amount of Cyt. c and AIF, the expressions of Bax and Bad, and down-regulation of the expressions of Bcl-2 and Bcl-xL. CONCLUSION: Tetracaine above 0.3125 g/L (1/32 of its clinical applied dosage) has a dose- and time-dependent cytotoxicity to HCEP cells in vitro, with inducing cell apoptosis via a death receptor-mediated mitochondrion-dependent pathway.     

Ceramide-induced apoptosis in rabbit corneal fibroblasts

PURPOSE: To evaluate the effect of various ceramides on the apoptosis of corneal fibroblasts and to determine the pathway on which they act. METHOD: Corneal fibroblasts isolated and cultured from New Zealand white rabbits were exposed to various concentrations of ceramide types II and VI and phytoceramide types II and VI, and their apoptotic response was evaluated using an LDH assay and Hoechst and Annexin V staining. Corneal fibroblasts were preincubated with various concentrations of the CPP32-like protease inhibitor Z-VAD-FMK, the caspase-8 inhibitor IETD-CHO, and the caspase-9 inhibitor Z-LEHD-FMK before treatment with ceramide, and apoptotic response was assayed by LDH assay. In addition, cells treated with ceramide or phytoceramide were stained with an antibody to cytochrome c. RESULTS: At concentrations of 20 microM and higher, all 4 ceramides increased fibroblast apoptotic response significantly after 12 hours. Hoechst staining showed shrinkage of the cytoplasm, formation of apoptotic bodies, and nuclear fragmentation after ceramide exposure, and Annexin V staining showed small vesicles around the cell membrane. The CPP32-like protease inhibitor reduced the apoptotic response to all 4 ceramides. The specific caspase-8 inhibitor reduced the apoptotic response to ceramide type VI and phytoceramide types II and VI, whereas the specific caspase-9 inhibitor significantly reduced the apoptotic response to phytoceramide types II and VI. Following exposure to ceramides, corneal fibroblasts stained positively with antibody to cytochrome c. CONCLUSION: Ceramide induced apoptosis in cultured corneal fibroblasts. This apoptosis involved the caspase cascade and the mitochondrial pathway.     

UVB irradiation-induced changes in the 27-kd heat shock protein (HSP27) in human corneal epithelial cells

PURPOSE: This study investigated the presence of the 27-kd heat shock protein (HSP27) and its responses to ultraviolet B (UVB) irradiation in human corneal epithelium and in cultured corneal epithelial cells. METHODS: Human corneal epithelial cells including presumed corneal epithelial stem cells were cultured in vitro. HSP27 expression and intracellular localization in normal corneas or cultured corneal cells were examined using immunofluorescence staining. The expression of HSP27 in cultured corneal cells was also detected using western blotting, and the phosphorylated isoforms of HSP27 were identified using isoelectric focusing. RESULTS: In normal corneal tissue, HSP27 was present in limbal basal and suprabasilar epithelial cells. In cultured epithelial corneal cells, HSP27 expression was heterogeneous: Some cells expressed virtually no HSP27 and others showed relatively strong expression. HSP27 was localized to the cytoplasm in nonstressed cells and translocated to the perinuclear and nuclear areas after UVB irradiation. UVB irradiation also induced the phosphorylation of HSP27, resulting in the increase in monophosphorylated isoform and formation of biphosphorylated isoform. UV induced the phosphorylation of HSP27 apparently through activation of p38 mitogen-activated protein kinase. CONCLUSION: HSP27 is present mainly as a nonphosphorylated isoform in corneal epithelium and cultured corneal epithelial cells under nonstressed conditions. The constitutional expression of HSP27 suggests that it plays a physiologic role in the cornea. After UVB irradiation, HSP27 undergoes rapid phosphorylation and translocation. This stress response may be related to a protective role of HSP27 for survival of UVB-exposed corneal cells.     

Caspase-independent component of retinal ganglion cell death, in vitro

PURPOSE: Although in vitro and in vivo models demonstrate caspase activation in retinal ganglion cells (RGCs) undergoing apoptosis, the caspase-independent component of RGC death is unclear. Identification of the precise mechanisms of cell death in these distinct neurons is essential for the development of effective neuroprotective strategies in glaucoma. Because TNF-alpha and hypoxia have been implicated in RGC death during glaucomatous optic nerve degeneration, this study was conducted to determine whether RGCs survive exposure to TNF-alpha or hypoxia in the presence of caspase inhibitor treatment, and whether mitochondrial dysfunction is involved in RGC death induced by these glaucomatous stimuli. METHODS: Primary cultures of rat RGCs were exposed to TNF-alpha or hypoxia for up to 48 hours. The temporal relationship of RGC death with the loss of mitochondrial membrane potential and the release of cell death mediators, including cytochrome c and apoptosis-inducing factor (AIF), was studied in the absence and presence of specific inhibitors of caspases. In addition, treatment with a free-radical scavenger, 4-hydroxytetramethylpiperidine-1-oxyl (tempol; 5 mM), was used in some experiments. Cell viability was assessed using calcein assay, and annexin V binding combined with propidium iodide staining was used for the distinction of apoptotic and necrotic cells. Caspase-3-like protease activity was measured using a fluorometric assay, and for the in situ detection of caspase activity, immunocytochemistry was performed with a cleavage-site-specific antibody. The time course of alterations in the mitochondrial membrane potential and the release of cell death mediators in individual cells undergoing cell death were assessed with a fluorescent tracer and subsequent immunocytochemistry. In addition, a fluorescent dye, dihydroethidium was used to assess the generation of reactive oxygen species (ROS). RESULTS: Findings of this study revealed that the loss of mitochondrial membrane potential and the release of cell death mediators accompanied RGC death induced by TNF-alpha or hypoxia. Although caspase inhibitor treatment temporarily decreased the rate of apoptosis, caspase inhibition was not adequate to block RGC death if the mitochondrial membrane potential was lost and mitochondrial mediators were released. Despite the inhibited caspase activity, survival rate was less than 70% after a 48-hour incubation with death stimuli, and both apoptotic and necrotic cells were detectable in these cultures. When combined with caspase inhibition, tempol reduced the production of ROS and provided an additional 20% increase in RGC survival. CONCLUSIONS: Based on these novel findings, RGC death induced by TNF-alpha or hypoxia involves a caspase-independent component, and reducing the free-radical generation provides additional protection of RGCs temporarily saved by caspase inhibition. Therefore, neuroprotective strategies in glaucoma should include tools to improve the ability of these neurons to survive the cytotoxic consequences of mitochondrial dysfunction.     

紫外线B照射对人晶状体上皮细胞质膜钙ATP酶3表达的影响

背景 细胞质膜钙ATP酶3（PMCA3）参与维持晶状体上皮细胞（LECs） Ca2＋的平衡,可能与白内障的病理过程有关,紫外线B（UVB）是引起白内障的重要因素之一,但UVB对LECs中PMCA3表达的影响少有报道. 目的 研究UVB对人LECs系B-3（HLE B-3） PMCA3表达的影响. 方法 对HLE B-3细胞进行培养和传代,当细胞达80％以上融合时分别暴露于用不同剂量（0、5、10、20 mJ·s/cm2）的UVB分别照射0、20、40和80 s,然后继续培养24、48和72 h,用MTT法检测不同剂量UVB照射后细胞的生存率;用JC-1染色法检测UVB照射后细胞线粒体膜电位（△ψm）的变化;以DCFH-DA染色法检测UVB照射后细胞内活性氧簇（ROS）的变化;采用annexin V-FITC/PI染色法检测UVB照射后细胞的凋亡情况;用Fluo-3/AM染色法检测细胞内Ca2＋浓度的变化;分别采用实时荧光定量PCR（real-time PCR）法和Western blot法检测UVB照射后细胞中PMCA3 mRNA及PMCA蛋白的表达变化. 结果 随着UVB照射剂量的增加和照射时间的延长,细胞生存率均明显下降,差异均有统计学意义（F分组=72.411,P=0.000; F时间=36.588,P=0.000）,其中10 mJ·s/cm2、20 mJ·s/cm2 UVB照射后24 h HLE B-3细胞的生存率分别为（75.3±2.2）％和（48.7±4.5）％,明显低于对照组的（100.0±0.0）％,差异均有统计学意义（P=0.001、0.000）;5、10、20 mJ·s/cm2 UVB照射后48 h细胞的生存率分别为（84.9±1.2）％、（69.3±17.4）％和（32.8±4.5）％,均明显低于对照组的（100.0±0.0）％,差异均有统计学意义（P=0.047、0.000、0.000）;5、10、20 mJ·s/cm2 UVB照射后72 h细胞的生存率分别为（55.1±3.0）％、（42.1±1.9）％和（26.1±4.7）％,与对照组的（100.0±0.0）％相比生存率明显降低,差异均有统计学意义（均P=0.000）.JC-1染色法检测表明,对照组细胞内可见红色荧光,5mJ·s/cm2 UVB照射后细胞内出现绿色荧光,10 mJ·s/cm2及20 mJ·s/cm2 UVB照射组出现绿色荧光增强,红色荧光减弱.5、10、20 mJ· s/cm2 UVB照射后细胞内的ROS由0.4％分别增加至35.8％、51.9％和76.7％.0 mJ·s/cm2 UVB照射组凋亡和坏死细胞比例为2.0％,5、10、20 mJ·s/cm2 UVB照射组凋亡和坏死细胞率分别为4.2％、7.6％和15.1％.10 mJ·s/cm2和20 mJ·s/cm2 UVB照射组细胞内Ca2＋水平分别为0 mJ·s/cm2照射组的（1.2±0.1）倍和（1.3±0.1）倍,差异均有统计学意义（P=0.039、0.004）.5、10和20 mJ·s/cm2 UVB照射组HLE B-3细胞PMCA3 mRNA表达量均明显低于对照组,差异均有统计学意义（P=0.001、0.004、0.000）,各组细胞中的PMCA蛋白相对表达量也均明显低于对照组,差异均有统计学意义（P=0.000、0.000、0.001）. 结论 UVB照射可导致白内障发生的机制可能与人LECs中PMCA3表达量下降和诱导LECs凋亡有关,UVB的作用呈剂量和时间依赖性.     

Cysteine starvation activates the redox-dependent mitochondrial permeability transition in retinal pigment epithelial cells

PURPOSE: Glutathione (GSH) plays a key role in protection against oxidative stress. L-cysteine is thought to be rate-limiting for the synthesis of glutathione (GSH) and therefore may be a critical component in protection against oxidative stress. The purpose of this study was to investigate the role of L-cysteine in GSH metabolism and oxidative stress in human retinal pigment epithelial (hRPE) cells. METHODS: To identify the role of cysteine in GSH metabolism in hRPE cells, a strategy of cysteine starvation was used to determine (1) GSH levels and oxidative stress by measuring reactive oxygen species (ROS) production, (2) mitochondrial membrane potential (Deltapsim) and mitochondrial ultrastructure by using conventional electron microscopy (EM), and (3) indices of cell viability and apoptosis including analysis of cells containing hypodiploid amounts of DNA. RESULTS: Cysteine starvation resulted in approximately a 95% decrease in GSH concentrations over 24 hours. The GSH Nernst redox potential (Eh) increased approximately 70 mV (Eh=-248 +/- 2.9 mV in control cells compared with Eh=-179 +/- 2.0 mV in cysteine-starved cells) indicating significant intracellular oxidation. Cysteine starvation increased the production of ROS by mitochondrial respiratory complex III (cytochrome bc1), determined using a pharmacological strategy that resulted in the loss of Deltapsim and cell death. The loss of Deltapsim and cell death was prevented with bongkrekic acid, an inhibitor of the adenine nucleotide translocator inhibitor, suggesting activation of the mitochondrial permeability transition (MPT). This conclusion was further supported by electron microscopic studies that showed significant mitochondrial swelling, a hallmark of MPT activation. Cell death was not prevented with either the broad-spectrum caspase inhibitor zVADfmk or the caspase 3-specific inhibitor DEVD-CHO, indicating that cytochrome bc1-mediated ROS production results in the MPT and necrosis. CONCLUSIONS: These results show that cysteine is a required component for normal GSH metabolism and protection against oxidative stress in hRPE cells.     

紫外线B照射在抑制人视网膜色素上皮细胞自噬中的作用

目的　探讨紫外线B(ultraviolet B,UVB)照射在抑制人视网膜色素上皮细胞株ARPE-19细胞自噬中的作用。方法　不同剂量UVB照射ARPE-19细胞后不同时间收集细胞,应用MTT法检测UVB照射对细胞存活的影响;Western blot检测细胞中自噬蛋白LC3的表达及LC3-II/LC3-I比率、Beclin-1蛋白表达水平;应用细胞自噬增强剂雷帕霉素（rapamycin,RAPA）和细胞自噬抑制剂氯化铵（NH₄Cl）进一步验证UVB照射对于细胞自噬的调节;采用环氧霉素（epoxomicin,EPO）检测泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)对于UVB作用的影响。MTT法检测细胞自噬变化对于细胞生存的影响。结果　UVB照射组诱导细胞剂量依赖性的死亡,100 mJ·cm^-2 UVB照射后24 h细胞出现明显死亡,和对照组相比差异具有统计学意义（P<0.05）;200 mJ·cm^-2 UVB照射后24 h和对照组相比差异有显著统计学意义(P<0.01);两种剂量的UVB照射后48 h细胞有一定程度的恢复。和对照组相比,UVB照射组ARPE-19细胞中LC3-II/LC3-I比率和Beclin-1蛋白表达水平均降低（均为P<0.05)。和UVB照射组相比,UVB+RAPA组ARPE-19细胞中明显升高了LC3-II/LC3-I比率,UVB+NH₄Cl组进一步降低了LC3-II/LC3-I比率,RAPA和NH₄Cl在一定程度上恢复了UVB照射降低的Beclin-1蛋白表达水平（均为P<0.05)。和对照组相比,EPO组明显增加了ARPE-19细胞中LC3-II/LC3-I比率、升高了Beclin-1蛋白表达水平（均为P<0.05)。与UVB照射组相比,UVB+EPO组ARPE-19细胞中LC3-II/LC3-I比率无明显改变(P>0.05),而Beclin-1蛋白表达水平差异有统计学意义(P<0.05)。与对照组相比,RAPA组细胞死亡率增多,两组间差异有统计学意义（P<0.05）,NH₄Cl组细胞无明显死亡,与UVB照射组相比,UVB+RAPA组和UVB+NH₄Cl组细胞死亡率均无明显改变（均为P>0.05）。细胞自噬的改变不能逆转UVB诱导的ARPE-19细胞死亡。结论　UVB照射抑制了ARPE-19细胞自噬的启动及自噬流,UPS的改变不影响UVB对于自噬流的抑制。UVB照射可通过减少RPE细胞、损害细胞自噬能力增加RPE细胞变性,增加年龄相关性黄斑变性发生的风险。     

人类永生化角膜内皮细胞的生物学特性

目的探讨人类永生化角膜内皮细胞在免疫反应中的分子表达及其刺激T淋巴细胞活化的能力。方法人类永生化角膜内皮细胞分别培养在含干扰素-γ（IFN-γ）（1000U／mL）和不包含IFN-γ（1000U／mL）的F99和M199培养液中,按照1：1等比例混合并含有5％小牛血清。健康志愿者采集外周血并分离外周血单核细胞（PBMCs）,应用锥虫蓝染色鉴定细胞的完整性和细胞数量。免疫组织化学染色法测定人类永生化角膜内皮细胞分别在有无IFN-γ诱导条件下的HLA—DP、DQ、DR抗原及CD40的表达。同时,体外共同培养人类永生化角膜内皮细胞和PBMCs,荧光活化细胞分类（FACS）法测定活化的T淋巴细胞中CD69的表达。结果IFN-γ能够诱导HLA—DP、DQ、DR抗原和CD40在人类永生化角膜内皮细胞上的阳性表达,表现为细胞膜和细胞质中的红色染色。CD3和CD28抗体预处理的外周血单核细胞中,20％的CD69阳性T淋巴细胞活化,无IFN-γ诱导时有10％阳性T淋巴细胞,而IFN-γ诱导组中,有12％的阳性T淋巴细胞,说明共同培养人类永生化角膜内皮细胞和PBMCs体系中,CD69阳性淋巴细胞数量增加。结论外周血中的T淋巴细胞可以被人类永生化角膜内皮细胞活化,由此人类永生化角膜内皮细胞是具有免疫学特性潜能的抗原递呈细胞。     

Mitochondrial dynamics in differentiating fiber cells of the mammalian lens.

The distribution of mitochondria was investigated in living rat and monkey lenses using rhodamine 123 staining and confocal microscopy. In both species, epithelial cells contained abundant small mitochondria scattered throughout the cell volume. Mid-sagittal slices of the living monkey lens revealed that, at the lens equator, mitochondria were only present in fiber cells to a depth of approximately 100 microns. Mitochondria were not present in fiber cells that had already reached the suture line or fibers abutting the central epithelium. Dual-staining with rhodamine 123 and the vital nuclear stain thiazole orange revealed that the loss of nuclei and mitochondria was coincident during fiber cell differentiation.     

Synthetic bile acid derivatives induce nonapoptotic death of human retinal pigment epithelial cells

PURPOSE. To study whether the synthetic ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) derivatives, which we have synthesized and have reported their apoptosis-inducing effect, have the effect on the proliferation of retinal pigment epithelial cells. METHODS. UDCA, CDCA, and their synthetic derivatives were administered in culture to the human retinal pigment cell line, ARPE-19. The effect on cell viability and growth was assessed by trypan blue dye exclusion. In order to evaluate the type of cell death, mitochondrial membrane potential assay, DNA electrophoresis, TUNEL assay, nuclear staining and Western blotting for caspase-3 and poly(ADP-ribose) polymerase (PARP) activities were conducted. RESULTS. Unlike UDCA and CDCA, which did not exhibit a significant effect on viability, their synthetic derivatives decreased the viability of ARPE-19 cells in a concentration-dependent manner. The cells treated with the synthetic derivatives did not demonstrate the characteristic findings of apoptosis, such as DNA ladder, DNA fragmentation, nuclear condensation or fragmentation, and caspase-3 and PARP activation. The reduction of mitochondrial membrane potential was shown. In electron microscopical study nuclear condensation was not shown. CONCLUSIONS. The synthetic UDCA and CDCA derivatives induced nonapoptotic death of ARPE-19 cells.