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.     

Inhibitive Effects of Quercetin on Rabbit Tenon Capsule Fibroblasts Proliferation

Purpose:To study the inhibitive effects of quercetin (QU) on the fibroblasts proliferation of rabbit Tenon's capsule and its mechanism. Methods: Cultured fibroblasts were exposed to different concentrations of QU solution and investigated by microculture tetrazolium (MTT) assay. The effect of QU was obser ved on cells cycle using the flow cytometer. Results: QU can suppress the proliferation of rabbit Tenon's capsule fibroblasts in vitro and show a dose-time dependent tendency. Flow cytometer results showed 26.92% cell increase in G1 phase, 23.50% decrease in S phase and 3.42% decrease in G2 phase. Conclusions: QU can suppress the proliferation of rabbit Tenon's capsule fibroblasts in vitro and show a dose-time dependent tendency. QU may effect all phase of cell cycle and inhibit cell proliferation by inhibiting Gl phase transitting to S phase and G2 phase. Eye Science 2005; 21:175-178.     

小檗胺抑制兔Tenon囊和滤过道内成纤维细胞增殖的实验研究

目的:研究钙调素拮抗剂小檗胺(berbamine,BER)对兔Tenon囊成纤维细胞和兔小梁切除术后滤过道内成纤维细胞增殖的抑制作用。方法:体外培养兔Tenon囊成纤维细胞,经不同浓度BER处理不同时间后,细胞计数Kit8(CCK8)法检测BER对兔Tenon囊成纤维细胞增殖的抑制作用,流式细胞仪检测其凋亡率及细胞周期的变化。HE染色检测BER对兔小梁切除术后滤过道内成纤维细胞增殖的抑制作用。结果:兔Tenon囊成纤维细胞经不同浓度BER处理不同时间后细胞增殖受到抑制,并呈时间剂量依赖性。流式细胞仪检测发现BER处理后兔Tenon囊成纤维细胞呈现G1期阻滞,细胞凋亡率明显增加,20mg/LBER处理9h,细胞凋亡率从0.64%增加到31.86%。HE染色显示BER显著抑制兔小梁切除术后滤过道内成纤维细胞的增殖。结论:小檗胺在体内外均能抑制成纤维细胞的增殖,其可能是通过诱导细胞凋亡方式抑制兔Tenon囊和滤过道内成纤维细胞的增殖。     

The influence of high molecular weight sodium hyaluronate (Healon) on the production of migration inhibitory factor

The effect of sodium hyaluronate on the production of migration inhibitory factor (MIF) was studied in a two step MIF-assay. High molecular weight sodium hyaluronate (100 micrograms/ml), added during the inductory step of the MIF-assay, inhibited the production of MIF. The inhibitory effect did not appear to be due to physical factors such as steric hindrance, which may prevent mitogen binding, since cells preactivated with phytohemagglutinin A (PHA) did not produce MIF when incubated in the presence of sodium hyaluronate. The inhibitory effect was still measurable when the sodium hyaluronate was added upto two hours after stimulation of the mononuclear cells with PHA. Inhibition was also found when the cells were preincubated with sodium hyaluronate, and washed prior to mitogen stimulation. Sodium hyaluronate could only be removed from the cells by incubation with hyaluronidase or by incubation of the cells for at least two hours in culture medium, whereafter the cells could be stimulated to the same extent as normal untreated cells to produce MIF. This inhibitory effect on cytokine production may explain the reduced inflammatory reactions found both in vivo and in vitro in the presence of sodium hyaluronate.     

双氯芬酸钠水分离对晶状体上皮细胞融合的影响

目的研究双氯芬酸钠水分离及联合转核技术对残留后囊膜上的晶状体上皮细胞（LECs）融合的影响。方法将猪眼后囊膜LECs进行体外原代培养,分为对照组、加药组（双氯芬酸钠水分离）、加药转核组（双氯芬酸钠水分离联合核旋转）,观察LECs不同时期的增生、分化、融合情况及各组细胞的融合时间。结果加药组和加药转核组的细胞融合时间较对照组延长,差异有统计学意义（P〈0.01）,加药组的细胞出现核固缩现象,加药转核组残留细胞团明显减少。结论双氯芬酸钠水分离能延缓后发性白内障（PCO）的发生,双氯芬酸钠水分离联合转核能有效降低PCO的发生率。     

Growth Inhibition,Induction of Apoptosis by Green Tea Constituent （—）—

Purpose: To evaluate effect of green tea extract (-)-Epigallocatechin-3-gallate (EGCG) in cultured rabbit lens epithelial cells in order to pave a new way to postcapsular opacity (PCO) prevention.Methods: Cell survival rate was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) coloimetric assay. Cell apoptosis was detected by electron microscopy, Hochest 33258 stain and flow cytometer. DNA fragment was detected using agarose gel electrophoresis.Result: Proliferation of the cultured rabbit lens epithelia cells was inhibited by EGCG in a dose and time dependent manner. Morphologic study showed that the cells became shrunk, round shaped with their nuclei condensed and broken. Apoptotic bodies were also seen under electron microscope and in Hochest 33258 stain assay 24 hours after EGCG was added to the medium. DNA ladders were shown in agarose gel eletrophoresis. In flow cytometry assay, apoptosis peak was also evident.Conclusion: Green Tea Constituent(-)-Epigallocatechin-3-g     

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.     

Inhibition of apoptosis and reduction of intracellular pH decrease in retinal neural cell cultures by a blocker of carbonic anhydrase

PURPOSE: Methylglyoxal and glyoxal are intermediates of advanced glycation end products (AGEs). These substances, as well as hydrogen peroxide, induce retinal neurons to reduce their intracellular pH and augment their production of reactive oxygen species, leading to apoptosis. Because these processes may play a role in diabetic retinopathy, the authors undertook this study to investigate the protective action of dorzolamide, an inhibitor of carbonic anhydrase, on retinal neural cells. METHODS: E1A-NR3 cells were incubated with varying concentrations of glyoxal, methylglyoxal, and H2O2 for different periods of time in the presence or absence of dorzolamide. Apoptotic changes were determined by cytofluorometry after the cells were incubated with appropriate dyes and antibodies. The parameters studied were DNA strand breaks (TUNEL assay), subdiploid DNA content (sub-G1 assay), annexin V binding, reactive oxygen species intermediates production, active caspase-3, N(epsilon)-(carboxymethyl)lysine (a glycation product), and intracellular pH. RESULTS: Optimal conditions for detection of the cell-protecting effect of dorzolamide were incubation with 0.6 to 0.8 mM glyoxal or methylglyoxal for 5 hours or with 0.1 mM H2O2 for 30 minutes, respectively, followed by 20-hour incubation with fresh medium. All apoptotic changes were reduced in the assays in which dorzolamide was included. CONCLUSIONS: Dorzolamide reduced the damage inflicted on retinal neural cells by agents that induced apoptosis and, therefore, can be considered a neuroprotectant.     

体外培养兔角膜内皮细胞电生理特性的初步研究

目的通过测定体外培养兔角膜内皮细胞(rabbitcorneal endothelial cells,RCECs)静息电位及全细胞电流,初步研究角膜内皮细胞的电生理特性。方法采用后弹力层及内皮层揭取联合组织块法进行RCECs体外培养,采用膜片钳全细胞记录模式记录原代及传代RCECs细胞静息电位及全细胞电流,绘制电流-电压(I/V)曲线。结果体外培养原代RCECs的平均静息膜电位水平为(20.9±1.7)mV(n=6),第2代RCECs的平均静息膜电位水平为(15.1±1.8)mV(n=6),两者差异有统计学意义(P<0.01)。在钳制电位+60mV下,原代RCECs全细胞电流为(396.2±39.0)pA(n=5),大于第2代RCECs的全细胞电流(201.4±45.2)pA(n=5),差异有非常显著的统计学意义(P<0.01)。结论后弹力层及内皮层揭取联合组织块法获得的原代及传代RCECs,在静息电位和全细胞电流等电生理特性方面存在差异。     

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。     

Inhibition of diclofenac formulated in hyaluronan on angiogenesis in vitro and its intraocular tolerance in the rabbit eye

Purpose: To investigate the effect of diclofenac, a potent nonsteroidal anti-inflammatory drug, formulated in hyaluronan (diclofenac/HA) on angiogenesis in vitro and its intraocular toxicity in vivo. Methods: The effect of diclofenac/HA on angiogenesis was determined by choriocapillary endothelial cells on Matrigel stimulated by vascular endothelial growth factor (VEGF). The tube areas were quantified by image digital analysis. For toxicity study, diclofenac/HA was injected intravitreally with a dose range from 100 to 1080 μg in 26 rabbits following gas compression vitrectomy. Potential toxicity was assessed by indirect ophthalmoscopy and by histological studies (light and electron microscopy). Retinal function was monitored by electroretinography (ERG) in six rabbits that received 400 μg of diclofenac/HA. Results: Diclofenac/HA, 180, 90 μg/ml, inhibited tube formation to 24% and 55% of the standard group (Media Ham’s F12 plus 5% fetal calf serum and 50 ng/ml VEGF) respectively (P<0.01). Intravitreal injection of 540 μg or higher doses of diclofenac/HA resulted in ocular toxicity in the rabbit, demonstrated as cataract, vitreous haze and retinal damage observed by indirect ophthalmoscopy and light- and electron- microscopic examinations. No toxicity was observed in the eyes that received 400 μg or less diclofenac/HA, which was further supported by the normal ERG examined at 4 and 25 days post injection. Conclusions: Diclofenac/HA inhibits tube formation in vitro and is nontoxic to the rabbit retina at concentrations that are inhibitory to tube formation. Our results suggest diclofenac/HA may be an effective candidate to inhibit ocular neovascularisation related to granulomatous reaction in the eye. Received: 28 April 1999 Revised: 23 August 1999 Accepted: 24 August 1999     

双氯芬酸钠滴眼液对培养人晶状体上皮细胞的抑制作用

目的　研究双氯芬酸钠滴眼液对人晶状体上皮细胞增殖的抑制作用。方法　分离培养人晶状体上皮细胞 ,在培养液内加入双氯芬酸钠滴眼液 ,并以高三尖杉酯碱作为对照。测定药物对细胞的半效抑制量 ;观察药物对细胞贴壁和形态学的影响。结果　双氯芬酸钠滴眼液作用 2 4h对晶状体上皮细胞半效量ID50 为 0 78μg/ml,72hID50 为0 63 μg/ml,作用时间对细胞的抑制率无明显差异 (P >0 0 5 ) ;与对照组相比 2 4h的ID50 无显著差异 (P >0 0 5 ) ,而 72hID50 有明显差异 (P <0 0 5 )。结论　双氯芬酸钠滴眼液对培养人晶状体上皮细胞在体外试验有抑制作用 ,但作为滴眼液能否作为防治后囊混浊的一种药物尚需进一步研究才能确定     

Release of endogenous ascorbic acid preserves extracellular dopamine in the mammalian retina.

PURPOSE: To investigate whether the inhibitory effect of nitric oxide (NO) on dopamine release from the retina is due to chemical oxidation of dopamine in the extracellular medium rather than to an inhibitory effect on dopamine release from retinal neurons. METHODS: Dopamine was incubated in Krebs bicarbonate medium and its rate of chemical degradation measured by high-performance liquid chromatography (HPLC). The effects of NO donors and antioxidants on dopamine were assessed by comparing dopamine degradation in the presence and absence of drug. The effects of NO donors on the K-evoked release of [3H]dopamine were measured from isolated superfused rabbit retinas. The release of ascorbic acid from the isolated rat retina and from an eyecup preparation in anesthetized rabbits was measured by HPLC. RESULTS: After 10 minutes' incubation in Krebs bicarbonate medium, the dopamine concentration decreased by 20%. This decline increased to 80% in the presence of S-nitroso-N-acetyl-DL-penicillamine (SNAP) or sodium nitroprusside (SNP). The increased rate of dopamine degradation was abolished if retina was incubated in the medium and then removed before the incubation of dopamine. The protective effect of preincubation with tissue was lost in the presence of ascorbate oxidase suggesting the release of ascorbic acid. HPLC analysis confirmed a substantial release of ascorbic acid from both rabbit and rat retinas. The K-evoked release of [3H]dopamine from the rabbit retina was inhibited by SNP. CONCLUSIONS: NO can rapidly, oxidize dopamine in physiological medium, but in the presence of retina, sufficient endogenous antioxidants (mainly ascorbate) are released to prevent this chemical reaction. Thus, the inhibitory action of NO on dopamine release results from an action on retinal neurons. Ascorbate release in the retina may have an important physiological role in prolonging the life of dopamine, which often has to diffuse long distances from axons in the inner plexiform layer to receptors in other retinal layers.     

Mast cell chymase induces conjunctival epithelial cell apoptosis by a mechanism involving degradation of fibronectin

PURPOSE: To determine the mechanism of conjunctival epithelial injury in vernal keratoconjunctivitis, we investigated the effects of human chymase on conjunctival epithelial cells in vitro. METHODS: Human conjunctival epithelial cells were incubated with human chymase for 24 or 48 hr at levels of activity that were likely to exist in the tear fluid of patients with vernal keratoconjunctivitis. Morphologic changes of the cells were observed by phase contrast microscopy. To determine the number of detached cells, we used an automated cell counter, while apoptotic cells were quantitated by flow cytometry. The level of soluble fibronectin in conditioned medium was measured by ELISA. RESULTS: Most of the cells in the incubation with chymase were detached by 24 hr. However, chymase-mediated apoptosis was a slower process and was only detected after incubation of cells with chymase for 36 to 48 hr. Both cell detachment and apoptosis were blocked when cells were incubated in fibronectin-coated plates. The increase of soluble fibronectin was dependent on the amount of chymase added and the exposure time. A caspase inhibitor (antiapoptotic agent) rescued cells from apoptosis but did not prevent cell detachment. These results indicate that chymase-induced apoptosis of conjunctival epithelial cells represents anoikis, which is a slowly occurring apoptotic process induced by lack of adhesion to an extracellular matrix. CONCLUSIONS: Human mast cell chymase caused conjunctival epithelial cell detachment by degrading fibronectin, and this led to secondary apoptosis.     

Mechanism of growth inhibitory effect of Mitomycin-C on cultured human retinal pigment epithelial cells: apoptosis and cell cycle arrest.

PURPOSE: To investigate the therapeutic potential of Mitomycin-C (MMC) in the management of proliferative vitreoretinopathy, the antiproliferative effect of MMC on cultured human retinal pigment epithelial (RPE) cells were investigated in vitro. METHODS: Drug sensitivities of cultured human RPE cells to MMC were determined using the tetrazolium dye assay. In order to detect the presence of apoptosis, DNA fragmentation was assessed by DAPI staining, and TdT-dUTP nick-end labeling (TUNEL) assay. The relative amount of DNA fragmentation was quantified by flow cytometric analysis. To analyze the cell cycle response of RPE cells to MMC, flow cytometric analysis of propidium iodide stained nuclei was performed. The levels of proteins related to DNA damage in the RPE cells were then determined by Western blot analysis. RESULTS: MMC inhibited cell proliferation in a dose-dependent manner. The majority of RPE cells following treatment with 10 microg/ml of MMC exhibited fragmented nuclei as observed by DAPI staining and TUNEL assay. Cell cycle analysis demonstrated an accumulation of cells arrested in S and G2/M phase following treatment with 1 microg/ml of MMC. At 10 microg/ml of MMC, a dramatic increase of the cell population in the sub G1 peak, which can be considered a marker of cell death by apoptosis, was observed by flow cytometry. Western blot analysis of p53 and p21 revealed a gradual increase in the level of these proteins when RPE cells were exposed to increasing concentrations of MMC. CONCLUSIONS: This study demonstrated that the response of RPE cells to MMC was bi-directional: 1) partial arrest of the cell cycle at S, G2/M phase, and 2) induction of apoptotic cell death.     

γ—干扰素及肝素抑制兔晶体上皮细胞生长的实验研究

目的:研究γ—干扰素及肝素对体外培养的兔晶体上皮细胞(RLEC)的生长抑制作用,探讨后发性白内障的防治方法。方法:RLEC原代细胞来自正常健康的新西兰大白兔。胰酶消化法传代。第三代细胞分别与0.1～10000u/ml剂量的γ—干扰素及100～5000u/ml的肝素孵育48小时,用MTT法检测γ—干扰素及肝素对晶体上皮细胞生长的抑制作用效果。结果:0.1～100u/ml剂量组的γ—干扰素对体外培养的RLEC无明显的抑制作用,1000u/ml及10000u/ml剂量组的γ—干扰素则对RLEC有较明显的抑制作用,其增殖抑制率分别为27％及38％。100～5000u/ml剂量组的肝素对RLEC均有明显的抑制作用。结论:一定浓度的γ—干扰素及肝素对兔晶体上皮细胞生长具有直接抑制作用。眼科学报1997;13:167—169。     

Caspase and proteasome activity during staurosporin-induced apoptosis in lens epithelial cells

PURPOSE: To determine what caspases are activated during staurosporin-induced apoptosis in cultured bovine lens epithelial cells (BLECs), to study the time course of caspase activation in relation to morphologic changes, and to investigate the effect of caspase and/or proteasome inhibition on apoptosis. METHODS: BLECs were incubated with staurosporin at different concentrations or for different times. Phosphatidylserine (PS) externalization was detected by annexin-V labeling, nuclear morphology was studied by staining with Hoechst 33342 stain (Hoechst, Frankfurt, Germany), and the percentage of apoptotic cells was determined by the TdT-dUTP terminal nick-end labeling (TUNEL) assay. The activity of caspase-1, -2, -3, -4, -8, and -9 as well as the chymotrypsin-like activity of the proteasome was measured by the use of fluorogenic peptide substrates. Inhibition of the proteasome was performed by incubation with 10 microM lactacystin, and caspases were inhibited by 1 microM Z-DEVD-FMK or 20 microM Z-VAD-FMK. RESULTS: Staurosporin treatment caused a dose- and time-dependent increase in the number of apoptotic cells and in caspase-3 activity. Activation of caspase-2, -4, -8, and -9 was also seen. Caspase activity was increased after 3 hours' incubation with 1 microM staurosporin, which is also the time when most cells became annexin-V-positive. Nuclear changes indicative of apoptosis, viewed with both Hoechst and TUNEL staining, appeared after 4 to 6 hours of staurosporin incubation. Incubation of BLECs with lactacystin caused reduction of proteasome activity and increased apoptosis, evidenced in both the TUNEL assay and caspase-3 activation. Preincubation of lens epithelial cells with caspase inhibitors caused complete inhibition of lactacystin- or staurosporin-induced caspase-3 activation (Z-DEVD-FMK/Z-VAD-FMK) and also of caspase-2, -4, -8, and -9 (Z-VAD-FMK), but the reduction in TUNEL-positive cells was only partial. PS translocation and DNA fragmentation after staurosporin treatment occurred despite complete caspase blockade. CONCLUSIONS: Staurosporin-induced apoptosis in BLECs involves activation of several caspases. Inhibition of the proteasome causes caspase-3 activation and apoptosis. Both staurosporin- and lactacystin-induced apoptosis can be executed in a caspase-independent manner. The present data are useful for understanding of proteolytic mechanisms during apoptosis in lens epithelial cells, which may be an important event in normal lens development as well as in some types of cataract.     

槲皮素对高糖培养牛视网膜周细胞增殖和凋亡的影响

目的:观察槲皮素(Quercetin,Que)对高糖状态下牛视网膜毛细血管周细胞(bovine retinal microvascular pericyte,BRPC)增殖和凋亡的影响。方法:用MTT比色法观察25,50,100μmol/LQue2,4,6d,对高糖(25mmol/L)培养BRPC增殖的影响;TUNEL法检测6d后Que对BRPC凋亡的影响。结果:高糖组较正常组吸光度值降低(P<0.01),槲皮素组较高糖组吸光度值升高(P<0.05),以高浓度槲皮素组吸光度值最高。TUNEL法检测显示:高糖状态下部分BRPC出现凋亡改变,高糖组较正常组凋亡率升高(P<0.01),槲皮素组较高糖组凋亡率降低(P<0.05),以高浓度槲皮素凋亡率最低。结论:Que可减轻高糖对BRPC增殖的抑制作用,减少高糖状态下BRPC的凋亡。