Protective effects of epigallocatechin gallate after UV irradiation in cultured human retinal pigment epithelial cells

PURPOSE: To evaluate the protective effects of Epigallocatechin gallate (EGCG) against UV irradiation in cultured human retinal pigment epithelial (RPE) cells. METHODS: UV irradiation was produced by a UV lamp for 30 seconds with an irradiance of 3.3 mW/cm2. After 5 minutes and 1 hour, we administered different concentrations of EGCG (0, 5, 10, 15, 25, 50, 100 uM). The cell count was determined under a microscope using a counting chamber and the cell activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: The cell count of cultured human RPE cells after UV irradiation was markedly increased in the EGCG administration group, compared with the non-administrated group. The cell activity of the cultured human RPE cells after UV irradiation was markedly increased in the EGCG administration group and was increased in a dose-dependent way as determined by the MTT assay. CONCLUSIONS: The administration of EGCG increased the cell count and the cell activity after UV irradiation in cultured human retinal pigment epithelial cells; this suggests that EGCG provided protection against UV damage in cultured human retinal pigmented epithelial cells.     

彗星法分析紫外线诱导的人晶状体上皮细胞DNA的损伤及抗氧化剂对DNA损伤的拮抗作用

目的探讨紫外线诱导的人晶状体上皮细胞（LEC）DNA损伤修复机制及抗氧化剂对DNA损伤的拮抗作用。方法采用照射剂量为0．0（对照组）及2．5、5．0、7．5、10．0mJ／cm^2（实验1～4组）的紫外线照射培养的人LEC,碱性彗星法（CA）分析人LEC DNA单链断裂（SSB）的程度和接受10．0mJ／cm^2紫外线照射后的自身修复情况,以及人LEC在10．0mJ／cm^2紫外线照射前后不同浓度抗氧化剂维生素C（VitC）、牛磺酸、超氧化物歧化酶（SOD）和表没食子儿茶素没食子酸酯（EGCG）对DNA损伤程度的影响。结果对照组与4个实验组DNA SSB程度呈上升趋势,5个组比较,差异有统计学意义（F=17．259,P〈0．01）;10．0mJ／cm^2紫外线诱导的人LEC的DNASSB后,其半修复时间为60min;紫外线照射前,加入抗氧化剂牛磺酸、SOD、EGCG,实验组和对照组比较差异有统计学意义（F值分别为6.591、13.542、4．626,P〈0．01）,VitC对照组和实验组比较差异无统计学意义（F=1．451,P〉0．05）;紫外线照射后,加入抗氧化剂VitC、牛磺酸、SOD、EGCG,实验组和对照组比较差异有统计学意义（F值分别为6．571、4．810、6．824、9．182,P〈0．01）;紫外线照射前加入4种抗氧化剂,其组间效应的差异有统计学意义（F=8．870,P〈0．01）,紫外线照射后加入4种抗氧化剂,其组间效应的差异无统计学意义（F=0．101,P〉0．05）。结论人LEC DNA的损伤程度与紫外线剂量呈线性关系;外源性VitC、牛磺酸、SOD、EGCG对紫外线照射诱导的人LEC DNA的损伤均有拈抗作用。紫外线照射前加入抗氧化剂的效应由强至弱依次为SOD、EGCG、牛磺酸、VitC,紫外线照射后加入4种抗氧化剂的效应差异无统计学意义。SOD和EGCG是高效抗氧化剂。     

Morphological observation on cell death and phagocytosis induced by ultraviolet irradiation in a cultured human lens epithelial cell line

The purpose of this study is to observe dynamic morphological changes induced by ultraviolet (UV) irradiation in a cultured human lens epithelial cell line using electron microscopy, cell viability staining, time-lapsed videography and immunohistochemistry. Human lens epithelial cell line SRA 01-04 was cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 20% fetal bovine serum. Subconfluent cells were irradiated under a bank of UV lamps, which emitted 275-400 nm radiation with a maximum at 310 nm. The UV intensity was 20 microW cm(-2)at dosages from 0 to 10 mJ cm(-2). Alterations in the morphology of the living cells were monitored and recorded with phase-contrast microscopy and time-lapsed videography. At different times, the cells were fixed and examined by transmission electron microscopy (TEM), diamidinophenolindole (DAPI) staining, and in situ immunohistochemistry using TdT-mediated dUTP-biotin nick end labeling (TUNEL). Cell viability was also assessed with crystal violet staining. At low doses of UV exposure (2-5 mJ cm(-2)), time-lapsed videography revealed definitive cell death that appeared to be primarily apoptotic. The dead cell debris was engulfed and phagocytosed by neighboring living cells. Phase-contrast microscopy and TEM demonstrated that, at UV 10 mJ cm(-2), the cells not only showed typical apoptosis such as nuclear membrane shrinkage, chromatin condensation, and fragmentation into apoptotic bodies, but also necrosis such as swelling of the nucleus and cell body, and disruption of the plasma membrane. In support, DNA staining and in situ immunohistochemical reactions in the UV irradiated cells were both positive. The phagocytotic process was also seen with TEM. UV irradiation thus appears to cause both apoptosis and necrosis in the cultured human lens epithelial cell line. Active migration and phagocytosis of the cells appear to be stimulated by UV-induced damage. These findings may also aid in the understanding of UV injury and repair mechanisms of lens epithelial cells in vivo.     

The effect of up- and downregulation of MnSOD enzyme on oxidative stress in human lens epithelial cells

PURPOSE: Gene knockouts serve as useful experimental models to investigate the role of antioxidant enzymes in protection against oxidative stress in the lens. In the absence of gene knockout animals for Mn-containing superoxide dismutase (MnSOD), the effect of this enzyme on oxidative stress was investigated in a human lens epithelial cell line (SRA 01/04) in which the enzyme was up- or downregulated by transfection with sense and antisense expression vectors for MnSOD. METHODS: Human lens epithelial cells (SRA 01/04) were transfected with plasmids containing sense and antisense human cDNA for MnSOD. The enzyme levels were determined by Western blot analysis and immunocytochemistry. The protective effect of the enzyme against the cytotoxicity of H(2)O(2) was evaluated by cell viability, DNA strand breaks, and morphologic changes observed by transmission electron microscopy. In addition, the protective effect of this enzyme against photochemically induced stress and UVB irradiation in cells was assessed by measuring the damage of cellular DNA. RESULTS: The MnSOD level in upregulated cells was three times higher than in downregulated cells, and the enzyme surrounded the nucleus. Cells with elevated enzyme levels were more resistant to the cytotoxic effect of H(2)O(2) with greater cell viability. MnSOD-deficient cells showed dramatic mitochondrial damage when exposed to 50 micro M H(2)O(2) for 1 hour. Similarly, oxidative challenge by H(2)O(2), photochemically generated reactive oxygen species, or UVB irradiation produced greater DNA strand breaks in MnSOD-deficient cells than in those in which the enzyme was upregulated. CONCLUSIONS: These findings demonstrate the protective effect of MnSOD in antioxidant defense of cultured lens epithelial cells. This approach to modulating the enzyme level in cultured cells provides a new experimental model for study of the role of antioxidant enzymes in the lens.     

The effects of sub-solar levels of UV-A and UV-B on rabbit corneal and lens epithelial cells

The purpose of this work was to establish whether exposing cultured rabbit corneal and lens epithelial cells to ultraviolet radiation equivalent to several hours under the sun would damage the cells. Confluent rabbit corneal epithelial cells were irradiated with broadband UV-A or UV-B, and confluent lens epithelial cells were irradiated with broadband UV-A. The maximum dose of UV-A was 6.3 J cm(-2) and that of UV-B was 0.60 J cm(-2). Damage to corneal epithelial cell was studied using the terminal deoxynucleotidyl transferase mediated dUTP-X nick end labeling (TUNEL) assay and damage to lens epithelial cell was studied using the single cell gel electrophoresis (comet) assay and trypan blue exclusion assay. Lipid peroxidation was assayed using the thiobarbituric acid reaction. Both UV-B and UV-A induced cell death in corneal epithelial cells with different latent periods. UV-A damage included cell death, decreased viability and increased lipid peroxidation of lens epithelial cell. In addition, UV irradiation of the corneal and lens epithelial cells decreased the activity of catalase to thirty to fifty percent of its original value, while the activities of glutathione peroxidase and superoxide dismutase did not decrease within experimental error. Thus, even sub-solar UV radiation can cause irreversible damage to corneal and lens epithelial cells.     

硫辛酸对人晶状体上皮细胞抗紫外线损伤作用的研究

目的探讨硫辛酸在人晶状体上皮细胞（LECs）中抗紫外线损伤的作用。方法对人LECs进行培养和传代,当细胞达80%以上融合时进行不同强度的紫外线照射,照射剂量分别为37、73、112mW/cm2。分别于紫外线照射前、照射中和照射后用0.5mmol/L硫辛酸孵育细胞2h作为实验组,不加硫辛酸的细胞作为对照组。照射后的LECs用常规方法继续培养48h后进行固定,质量分数0.4%龙胆紫染色,自然风干后进行吸光度（A）值测定,计算细胞存活率并进行统计分析。结果正常培养的LECs24h完全贴壁,2～3d时70%以上的LECs达到融合。未用硫辛酸孵育的LECs组细胞连接疏松,细胞膜皱缩,可见部分漂浮细胞。硫辛酸孵育的LECs形态改变程度较轻。不同照射剂量的紫外线照射条件下各硫辛酸培养组LECs存活率差异有统计学意义（Fgroup=23.27,P〈0.01;Fdose=1460.34,P〈0.01）。紫外线照射的不同时间添加硫辛酸各组中LECs的生存率明显高于不添加硫辛酸组（P〈0.05）,而在紫外线照射剂量为半数致死量时其增强细胞存活率的作用最强。在照射剂量为半数致死量及1.5倍半数致死量时其细胞存活率依次为：照射后硫辛酸组〉照射前硫辛酸组〉照射中硫辛酸组〉对照组。结论硫辛酸可以提高紫外线照射后的人LECs的存活率。提示硫辛酸可以减轻紫外线对人LECs的光损害。     

Ultraviolet transmittance of human limbal epithelial cells cultured on human amniotic membranes

PURPOSE: To evaluate ultraviolet (UV) A and B transmittance by human limbal epithelial cells cultured on human amniotic membranes. METHODS: Human limbal epithelial cells were taken from the limbus of donor corneas and were cultured on human amniotic membranes with inactivated 3T3 fibroblasts for 2 to 4 weeks. Then, the cultured cells were examined histologically. Next, cells from different culture periods were irradiated with UV-A (365 nm) or UV-B (302 nm) at energy levels ranging from 50 to 800 microW/cm2, and UV transmittance was measured with a UV light meter. RESULTS: Histological examination revealed a monolayer of corneal epithelial cells on the amniotic membrane after 2 weeks of culture, and a layer of 3-4 cells was formed after 4 weeks. Transmittance of UV-A and UV-B was highest by the amniotic membrane alone, followed in decreasing order by limbal epithelial cells cultured on amniotic membranes for 2 weeks, 3 weeks, and 4 weeks. CONCLUSIONS: These results indicate that UV absorbance increases in proportion to the number of limbal epithelial cell layers in cultures on amniotic membranes. Limbal epithelial cells may need to be cultured until 3-4 layers are formed in order to prevent ocular damage by UV light after transplantation.     

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     

EGCG抗兔晶状体上皮细胞增殖机制中p38激酶作用的研究

目的:研究p38激酶在绿茶提取物中的表没食子儿茶素没食子酸酯[(-)-Epigallocatechin-3-gallate,EGCG]抗晶状体上皮细胞增殖机制中的作用。 方法:用噻唑蓝比色法(MTT比色法)研究p38的特异性抑制剂SB203580对EGCG抑制晶状体上皮细胞增殖作用的影响;用Western Blot法研究EGCG对p38激酶的磷酸化和非磷酸化水平的影响。 结果:(1)预先加入25μmol/L,50μmol/L的SB203580孵育1h后,100μmol/L EGCG对晶状体上皮细胞增殖的抑制率低于对照组,但这种差别没有统计学上的意义(P>0.05);200μmol/L EGCG组对晶状体上皮细胞的抑制率低于对照组,有统计学的意义(P<0.05);(2)在晶状体上皮细胞,磷酸化的p38基础水平很低。p38的磷酸化水平随EGCG浓度的增加逐渐增加,而非磷酸化的p38的水平与基础水平一样保持不变。以200μmol/L EGCG作用15min来研究其对p38的磷酸化和非磷酸化影响的时-效规律发现,加药后初期,p38的磷酸化水平很高,随后逐渐下降。同时,非磷酸化p38的水平保持不变。 结论:(1)EGCG对晶状体上皮细胞的增殖抑制作用可能通过p38通路;(2)EGCG对p38影响主要在于调节蛋白激酶的磷酸化与去磷酸化水平,不影响总蛋白含量。眼科学报 2003;19:236-238。     

Carteolol hydrochloride protects human corneal epithelial cells from UVB-induced damage in vitro

PURPOSE: To investigate whether carteolol hydrochloride has protective effects against ultraviolet B (UVB)-induced damage in human corneal epithelial cells (HCECs). METHODS: Cultured HCECs were exposed to a single dose of UVB 300 mJ/cm, and the cell viability was measured 12 hours after the UVB irradiation using a cell-counting kit. Test samples at 0.01-1.0 mmol/L (carteolol hydrochloride, timolol maleate, betaxolol hydrochloride, levobunolol hydrochloride, or nipradilol) were added to the HCECs before, during, or after UVB irradiation. UV absorption spectra for each drug sample were determined using a spectrophotometer. Hydrogen peroxide (H2O2) and carteolol hydrochloride were simultaneously added to the HCECs for 10 minutes, and the cell viability was measured 12 hours later. The ability of carteolol hydrochloride to scavenge superoxide anion (O2) and singlet oxygen (O2) was investigated using the MCLA chemiluminescence method. RESULTS: UVB irradiation decreased the number of viable HCECs in a dose-dependent manner. Carteolol hydrochloride at 1 mmol/L attenuated the UVB-induced cell damage when added before, during, or after UVB irradiation (P<0.01). Levobunolol hydrochloride at 1 mmol/L (P<0.01) added during or after irradiation and timolol maleate at 0.1 mmol/L or higher (P<0.05) added during irradiation attenuated the UVB-induced cell damage. Betaxolol hydrochloride and nipradilol had no effect. The UV absorption spectra of timolol maleate and levobunolol hydrochloride overlapped with the UVB wavelength spectrum, while carteolol hydrochloride, betaxolol hydrochloride, and nipradilol showed a partial overlap. Carteolol hydrochloride at 1 mmol/L (P<0.05) significantly inhibited H2O2-induced cell damage and was able to scavenge O2 (EC50 value: 48 mmol/L). CONCLUSIONS: These data strongly suggest that carteolol hydrochloride has a protective action against UVB-induced HCEC damage, and its radical scavenging ability may be an important basis for this effect.     

DNA repair and survival in human lens epithelial cells with extended lifespan.

PURPOSE: Ultraviolet-B radiation (290-320 nm) produces cataracts in animals and has been associated with human cataract formation in several epidemiological studies. UVB radiation decreases the long-term cell survival and changes the pattern of protein synthesis in cultured lens epithelial cells. However, the relationship between DNA photoproduct formation and long term cell survival in human lens epithelial cells is not known. In the present work, we used human lens epithelial cells with extended lifespan (HLE B-3 cells) to examine the kinetics of DNA repair and cell survival after UVB exposure. METHODS: Cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts were analyzed by radioimmunoassay. Long-term survival of the cells was determined by measuring their ability form colonies when plated at low density. RESULTS: HLE B-3 cells were repair competent after UVB (302 nm) exposure. Excision repair of the (6-4) photoproduct was more efficient than that of the cyclobutane dimer. Ninety five percent of the (6-4) photoproducts were repaired 24 h after 400 J/m2 UVB exposure, whereas 50% of the cyclobutane dimers were repaired during this time. When cells were split for the clonogenic assay immediately after irradiation, only 10% of the cells formed colonies following 7 days of culture in the serum-containing medium. When cells were split for the clonogenic assay after a 48 hour incubation in serum-containing medium, the colony-forming ability of the irradiated cells increased to 60% following culture in a serum-containing medium. CONCLUSIONS: These results indicate a close correlation between the repair of cyclobutane dimers and the increase in the long-term survival of the cells as measured by their colony-forming ability. The extended lifespan human lens epithelial cells HLE B-3 may be a useful model to investigate the mechanism and regulation of UVB-induced DNA repair in human lens cells.     

表没食子酸酯对高糖诱导的人晶状体上皮细胞氧化应激的影响

目的：：探讨表没食子酸酯( EGCG)对高糖诱导的人晶状体上皮细胞( human lens epithelial cells, HLEC )氧化应激的影响。方法：建立高糖诱导的HLEC氧化损伤模型,用不同浓度的EGCG干预,MTT检测细胞活力,倒置显微镜观察细胞形态,Hoechst-PI染色观察细胞凋亡,分光光度计检测上清液中超氧化物歧化酶( SOD )、谷胱甘肽过氧化物酶( GSH-Px)及丙二醛( MDA)的含量。结果：MTT 结果显示用10μmol/L EGCG 和100μmol/L EGCG处理后,HLEC活性分别提高到50.33%±3.52%和63.33%±4.63%,与氧化损伤组(32.67%±3.10%)比较差异具有统计学意义( P<0.05);EGCG干预的高糖条件下的HLEC较好地保持了细胞的形态,凋亡细胞数量减少,细胞内SOD、GSH-Px水平升高,MDA水平下降。结论：EGCG可能通过提高细胞内SOD、GSH-Px含量,降低MDA含量发挥其较强的抗氧化作用,从而为寻求有效的防治白内障药物提供可靠的实验依据。     

UV absorption in human oral mucosal epithelial sheets for ocular surface reconstruction

BACKGROUND: Ocular surface reconstruction with autologous oral mucosal epithelium has attracted attention as a novel treatment strategy that avoids allograft rejection. OBJECTIVES: To evaluate the absorption of ultraviolet (UV) A or B irradiation by human oral mucosal epithelium cultured on human amniotic membrane. METHODS: Human oral mucosal and limbal epithelial cells were co-cultured on amniotic membrane with inactivated 3T3 fibroblasts. The cell sheets were also subjected to UV-A (365 nm) or UV-B (302 nm) irradiation at energy levels ranging from 50 to 800 microW/cm2, and the UV absorption rate was measured with a UV irradiation meter. RESULTS: Cultured oral mucosal epithelium had a structure with 3-5 layers of cells, consistent with the histological features of cultured corneal limbal epithelium after 4 weeks. The decrease in UV-A absorption of cultivated oral mucosal epithelium ranged from 25 to 36% of that for cultured corneal epithelium. The increase in UV-B absorption by cultured oral mucosal epithelium between 200 and 800 microW/cm2 was approximately 145% of that for cultured corneal limbal epithelium. CONCLUSION: Our data demonstrated that cultured oral mucosal epithelium has low UV-A and high UV-B absorption capacity as compared with those of cultured corneal epithelium, suggesting that oral mucosal epithelium can compensate for UV absorption of corneal epithelium.     

Effects of yellow intraocular lenses on light-induced upregulation of vascular endothelial growth factor

PURPOSE: To investigate the protective effect of a blue-light filtering intraocular lens (yellow IOL) (YA60BB, Hoya) and an ultraviolet (UV)-absorbing IOL (VA60BB, Hoya) on light-induced phototoxicity to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore A2E and on the production of vascular endothelial growth factor (VEGF) after light exposure. SETTING: University of Tokyo, Tokyo, Japan. METHODS: The A2E-laden ARPE-19 cells were exposed to white light and a UV-absorbing IOL or a blue-light filtering IOL was placed over the light beam. After 48 hours of irradiation, the viability of the cells was determined with WST-1 (a sodium salt of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay, and the secreted protein level of VEGF was determined by enzyme-linked immunosorbent assay. RESULTS: Without an IOL, the white-light exposure decreased cell viability to 28% of the nonirradiated control. Although the UV-absorbing IOL tended to reduce light-induced cell death, the decrease was not significant. However, the presence of the blue-light filtering IOL significantly attenuated light-induced cell damage, increasing cell viability to 42%. The secreted VEGF protein level increased 3.2-fold after the A2E-laden RPE cells were exposed to white light. In the presence of the UV-absorbing IOL, the VEGF protein level decreased, but not significantly. The presence of the blue-light filtering IOL significantly attenuated the upregulated VEGF expression compared to upregulation without an IOL. CONCLUSION: This study supports the theory that a blue-light filtering IOL may be more protective against A2E-induced photochemical damage and inhibit more light-induced VEGF production than a conventional UV-absorbing IOL.     

Cellular death mediated by nuclear factor kappa B (NF-kappaB) translocation in cultured human lens epithelial cells after ultraviolet-B irradiation

PURPOSE: To determine the role of nuclear factor kappa B (NF-kappaB) in the death of lens epithelial cells (LECs) after ultraviolet (UV) irradiation. SETTING: Department of Ophthalmology, Ilsan Paik Hospital, Inje University, Korea. METHODS: Cultures of simian virus 40 transfected human LECs (HLE B-3 cells) were were irradiated with a UVB source (312 nm) located 10 cm from the bottom of the slides for 1, 2, 3, or 4 minutes. Cytotoxicity was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide method. Translocation of NF-kappaB was examined by immunocytochemistry using anti-NF-kappaB p65 antibody and electrophoretic mobility shift assay (EMSA). Sulfasalazine, a specific NF-kappaB inhibitor, was used to confirm the role of NF-kappaB by pretreating samples for 30 minutes before UV irradiation, after which cytotoxicity and NF-kappaB translocation were evaluated. RESULTS: When HLE B-3 cells were irradiated with UVB, translocation of NF-kappaB was observed with immunocytochemistry. These translocations peaked during EMSA 6 hours after UV irradiation. In HLE B-3 cells pretreated with sulfasalazine, the translocation of NF-kappaB was blocked. Cellular death after UV irradiation was also markedly reduced by sulfasalazine pretreatment. Ultraviolet irradiation can translocate NF-kappaB, and sulfasalazine is a useful blocking agent in this pathway. In this experimental model, sulfasalazine prevented cellular death after UV irradiation. CONCLUSION: The findings suggest that NF-kappaB plays an important role in cellular death after UV irradiation.     

白内障术后后囊浑浊的防治和抗人晶状体上皮细胞单克隆抗体的制备

目的制备可特异性识别人晶状体上皮细胞的单克隆抗体，筛选特异性强亲和力高的克隆。方法以原代培养人晶状体上皮细胞作为免疫原，使用杂交瘤技术制备抗人晶状体上皮细胞单克隆抗体（monoclonal antibody，McAb）。小鼠腹腔接种杂交瘤细胞制备腹水，用免疫荧光、免疫组化法对其特性进行鉴定。结果获得了1株稳定分泌抗人晶状体上皮细胞McAb的杂交瘤细胞系，其亚类为IgM。免疫荧光和免疫组化检测结果显示，此McAb与人晶状体上皮细胞膜上的抗原反应，与人眼其他组织呈阴性反应。结论所获抗体可特异性识别人晶状体上皮细胞，可进一步制备免疫导向药物，用于白内障术后后囊浑浊的治疗。     

Human lens cells in culture. I. Isolation of adult lens epithelial cells from lens capsule preparations and reactivation of nucleus-containing fiber cells

BACKGROUND: Bovine lens epithelial cells in culture revealed a high sensitivity against micromolar concentrations of linoleic acid. To prove the assumption that unsaturated free fatty acids are risk factors for cataractogenesis, human lens cell lines are needed. Furthermore, the reactivation of nucleus-containing fiber cells to mitotic growth may hint at their role in after cataract genesis. MATERIAL AND METHODS: Epithelium-capsule-preparations obtained by capsulorhexis were cultured in serum containing medium. Subculturing of these adult human lens epithelial cells was done by trypsinization. Fiber cell bundles from the equator region of a fetal human lens were transferred into culture medium. Aggregates of nucleus containing fiber cells were isolated from floating fiber cell bundles by trypsinization. Subculturing and cryoconservation of suitable cell lines. RESULTS: Primary culture of epithelium-capsule-preparations results in flattening, migration and proliferation of adult human lens epithelial cells. Nucleus containing fiber cells were reactivated to mitotic growth after adhesion to a suitable substratum. Established cell lines were received from adult human lens epithelial cells and fetal human fiber cells after repeated subculturing. CONCLUSIONS: Lens-capsule-preparations available from cataract surgery are well suited for the isolation of human lens cell lines, which were needed for testing cytotoxicity of drugs and for tracing of cataractogenic risk factors. The finding that nucleus containing fiber cells from the equator of human lenses can be reactivated to proliferating cells let us suppose, that these cells, which can not be removed easily from the posterior lens capsule, contribute to the after cataract formation.     

Mechanisms of apoptosis on human lens epithelium after ultraviolet light exposure

Purpose

The purpose of this study is to understand the mechanism of apoptosis occurring on a cultured human lens epithelial cell line after exposure to ultraviolet (UV) light. We intended to confirm the presence of cellular toxicity and apoptosis and to reveal the roles of p53, caspase 3 and NOXA in these processes.

Methods

Cells were irradiated with an ultraviolet lamp. Cellular toxicity was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining and fluorescent anti-caspase 3 antibodies were used for apoptosis investigation. The quantities of p53, caspase 3, and NOXA were measured by Western blotting for to investigate the apoptosis pathway.

Results

Cellular toxicity on the human lens epithelium markedly increased with time after UV exposure. On Hoechst staining, we found that apoptosis also remarkably increased after exposure to ultraviolet light, compared with a control group. In the immunochemical study using anti-caspase 3 antibodies, active caspase 3 significantly increased after exposure to ultraviolet light. On Western blotting, p53 decreased, while caspase 3 and NOXA increased.

Conclusions

Exposure of cultured human lens epithelial cell lines to ultraviolet light induces apoptosis, which promotes the expression of NOXA and caspase 3 increases without increasing p53. This may suggest that UV induced apoptosis is caused by a p53-independent pathway in human lens epithelial cells.     

Effect of eyelid closure and overnight contact lens wear on viability of surface epithelial cells in rabbit cornea.

PURPOSE: To determine the effects of open, closed eye, and overnight contact lens wear on homeostatic epithelial surface cell death in the rabbit cornea. METHODS: One eye of each rabbit was either closed by eyelid suture or fitted with one of the following test contact lenses: (1) low Dk/t rigid gas permeable (RGP) lens, (2) hyper Dk/t RGP lens, (3) hyper Dk/t soft lens. The other eye served as a control. After 24 hours, whole corneas were carefully excised and immediately stained with a calcein-acetoxymethyl ester-ethidium homodimer viability assay to quantify the number of nonviable surface epithelial cells. In addition, exfoliated corneal epithelial cells were collected with an eye irrigation chamber to determine cell viability. RESULTS: In the normal cornea, open-eye conditions showed significantly more nonviable surface cells in the central cornea than in the periphery (p < 0.05). Overnight wear of all test lenses and eyelid closure induced significant decreases in the number of nonviable cells on the central corneal surface compared with controls (p < 0.05). All exfoliated corneal epithelial cells collected by eye irrigation were nonviable. CONCLUSION: In the rabbit model, overnight contact lens wear significantly downregulated spontaneous epithelial surface cell death independent of lens rigidity or material oxygen transmissibility. These effects were similar to eyelid closure without lens wear. Taken together, these results suggest that eyelid closure and the physical presence of the contact lens may protect against the shear stress forces exerted by eyelid blinking, which are believed to cause central surface cell death and subsequent exfoliation.