Concentration dependent effects of hydrogen peroxide on lens epithelial cells

AIMS: To evaluate the effects of hydrogen peroxide exposure on the survival and proliferation of cultured lens epithelial cells. METHODS: TOTL-86 cells, a line of rabbit lens epithelial cells, were used. The survival and proliferation of TOTL-86 cells were quantified by a rapid colorimetric assay (MTT assay). To determine the effects of hydrogen peroxide, TOTL-86 cells were exposed to different concentrations of hydrogen peroxide. To determine the effect of cell numbers on the survival and proliferation of TOTL-86 cells at a fixed concentration of hydrogen peroxide, different numbers of cells were plated and exposed to hydrogen peroxide. To determine whether there is a synergistic effect between hydrogen peroxide and EGF, bFGF, PDGF-AA, and insulin, TOTL-86 cells were exposed to hydrogen peroxide combined with one of these growth factors. RESULTS: High levels (1 mM) of hydrogen peroxide killed TOTL-86 cells and sublethal levels (100 microM) suppressed their proliferation. From 1 nM to 1 microM of hydrogen peroxide, there was a dose dependent increase in the cell numbers. The initial seeded cell number dramatically affected the response to hydrogen peroxide. Although growth factors showed no synergistic effects with hydrogen peroxide on proliferation, both EGF and insulin, but not bFGF or PDGF, rescued TOTL-86 cells from the sublethal effect. CONCLUSION: Hydrogen peroxide in cooperation with some growth factors plays an important role in the proliferation of lens epithelial cell.     

葡萄糖浓度对培养的兔晶状体上皮细胞的影响

目的：观察不同浓度葡萄糖培养基对培养的兔晶状体上皮细胞形态、生长状态、细胞分化及TGF-β 2表达的影响.方法：以4.5,9,18和36g/L葡萄糖培养基处理原代培养的第2代兔晶状体上皮细胞,MTT法检测细胞生长状态,免疫组化法检测TGF-β2,α-SMA及PCNA.结果：相对于4.5g/L葡萄糖培养基,高浓度葡萄糖培养基可增强晶状体上皮细胞α-SMA与TGF-β 2的表达,而PCNA的表达无明显差异;MTT所反映的细胞生长状态呈下降趋势.结论：高浓度葡萄糖培养基可促进晶状体上皮细胞分化,对细胞的增殖有抑制作用,在此过程中TGF-β 2可能发挥着重要的作用.     

Susceptibility of lens epithelial membrane SH groups to hydrogen peroxide

Although membrane SH groups are thought to be targets of oxidative insults, no measurement of lens epithelial membrane SH groups following exposure to potentially damaging oxidants has been reported. Here we investigate the effect of hydrogen peroxide, an oxidant found in the aqueous humor, and of p-chloromercuriphenylsulfonic acid (p-chloromecuribenzene-sulfonic acid) (PCMBS), a relatively impermeant sulfhydryl probe, on membrane SH groups and ion homeostasis in cultured lens epithelial cells. Exposure to PCMBS caused a 10% loss of membrane SH groups, an increase in sodium and calcium levels, and a decrease in potassium, but did not affect the intracellular level of glutathione (GSH). After 5 min of exposure to an initial concentration of 1.0 mM hydrogen peroxide, GSH declined from 14.1 mM to 3 mM, there was a 20% loss of membrane SH groups and within 1 hr, potassium declined from 132 to 116 mM. Cells that were exposed to 0.1 or 0.5 mM peroxide did not exhibit significant loss of membrane SH groups and did not show a decrease in GSH comparable to that found in cells treated with 1 mM peroxide. The peroxide induced loss of membrane SH groups and subsequent change in ion homeostasis occurred only when there was a rapid and sustained loss of intracellular glutathione. Thus lens epithelial cell membrane SH groups are not only important in ion regulation but are targets of hydrogen peroxide when the intracellular level of GSH is significantly diminished.     

过氧化氢对人晶状体上皮细胞增生及VEGF表达的影响

目的:研究不同浓度的过氧化氢(H2O2)对体外培养的人晶状体上皮细胞增生及VEGF表达的影响。方法:体外培养的人晶状体上皮细胞系SRA01/04,在培养液中分别加入浓度为1,10,100nmol/L;1,10,100μmol/L和1mmol/L的过氧化氢处理40min,对照组无过氧化氢,随后在正常培养液中继续培养,在0,6,12和24h用MTT方法检测晶状体上皮细胞的增生活力;在24h用ELISA方法检测培养液上清中血管内皮生长因子(VEGF)的含量。结果:在培养液中加入1nmol/L~10μmol/L浓度的H2O2处理组,晶状体上皮细胞增生率(A值)随浓度较对照组明显增加,其中以10nmol/L最为明显;而100μmol/L,1mmol/L处理组,晶状体上皮细胞出现生长抑制。ELISA检测结果显示,1nmol/L~10μmol/L的过氧化氢刺激后,VEGF水平均高于对照组,且呈浓度依赖性,峰值出现在10nmol/L和10μmol/LH2O2处理组。结论:低浓度(1nmol/L~10μmol/L)的H2O2可以促进晶状体上皮细胞增殖,并使VEGF分泌增加;提示VEGF可能作为一种氧化应激产物,对晶状体上皮细胞起到保护、免受损伤的作用。     

过氧化氢对人晶状体上皮细胞内细胞质膜微囊蛋白的影响

目的探讨过氧化氢（H2O2）对人晶状体上皮细胞的细胞质膜微囊蛋白（CP）及磷酸化CP-1分布与表达的影响。方法给予人晶状体上皮细胞（SRA01／04）不同浓度及不同时间点的H2O2刺激。用激光共聚焦显微镜和免疫荧光显微镜观察CP及磷酸化CP-1的分布。通过免疫印迹试验观察CP表达水平的变化以及磷酸化CP—1的表达。结果通过激光共聚焦显微镜和荧光显微镜,观察到人晶状体上皮细胞的质膜和细胞质内含有丰富的CP;当给予细胞H2O2刺激后,细胞质内CP的分布增多;当刺激时间达到1h,细胞膜被破坏,但仍可观察到CP的分布情况。此外,H2O2的刺激可以使CP-1发生磷酸化。免疫印迹试验发现,随着H2O2作用时间的延长和刺激浓度的升高,细胞质膜和细胞总蛋白质的CP表达水平呈下调趋势。结论H2O2刺激人晶状体上皮细胞后,CP重新分布并可能破坏了细胞质膜微囊（caveolae）的结构,使得细胞的CP表达下调。细胞质膜微囊和CP可能在人晶状体上皮细胞中具有重要作用。     

H2O2对小窝蛋白在人晶状体上皮细胞中分布与表达的影响

目的 观察以H2O2刺激晶状体上皮细胞（LECs）后对细胞活性的影响及小窝蛋白（caveolin）在LECs膜及胞浆内的分布和表达量的改变。方法 用lmmol／LH2O2刺激LECs不同时间，MTT法观察细胞活性；激光共聚焦显微镜观察caveolin在细胞膜和胞浆内的分布；Western—blot法检测caveolin表达量的变化。结果 H2O2作用时间越长，细胞活性越低；对照组与试验组的差异有显著统计学意义。激光共聚焦显微镜观察到人LECs有丰富的caveolin；无刺激时主要分布在膜上；刺激后在膜及胞浆内的分布增多。Western-blot法发现H2O2作用后，caveolin的表达量减少。结论 H2O2对人LECs的活性产生影响，并导致细胞caveolin重新分布、表达量减少。     

晶状体上皮细胞凋亡与过氧化氢诱导白内障形成研究

目的 探讨晶状体上皮细胞凋亡与皮质性白内障形成的关系。方法用200μmol／L过氧化氢(H2O2)诱导离体兔晶状体白内障形成,TUNEL法检测H：0：作用1,6,18,24,48h的晶状体上皮凋亡细胞,同时测定晶状体超氧化物歧化酶(SOD)活性。结果H2O2作用1h,晶状体保持透明,未发现凋亡上皮细胞。随着作用时间的延长,凋亡上皮细胞数量逐渐增多,晶状体逐渐变混浊。SOD活性早期无变化,18h后逐渐降低。结论晶状体上皮细胞凋亡是皮质性白内障形成的细胞学基础,其发生先于晶状体抗氧化机制的变化。     

热休克预处理对晶状体上皮细胞过氧化氢氧化损伤的保护作用

目的热休克预处理诱导的热休克蛋白27对晶状体上皮细胞的保护作用,并初步探讨其机制。方法体外培养人晶状体上皮细胞株HLB3细胞,分为正常对照组、氧化损伤组和热休克处理组,观察过氧化氢(H2O2)处理后各组细胞活力、SOD、CAT的改变情况;AnnexinⅤFITC流式细胞仪检测晶状体上皮细胞凋亡;RTPCR检测各组中HSP27及p21基因的表达。结果热休克预处理可诱导HSP27产生,显著增加细胞活力0.27±0.04(与氧化损伤组0.24±0.02相比P<0.05),细胞凋亡率明显降低(8.34±1.19)%(与氧化损伤组15.69%±1.54%比较P<0.05),保护超氧化物岐化酶0.61±0.07(与氧化损伤组0.41±0.08比较P<0.05)、过氧化氢酶活性0.99±0.14(与氧化损伤组0.33±0.06相比P<0.05),降低p21mRNA的表达。结论热休克预处理可能通过诱导HSP27的表达保护氧化损伤的晶状体上皮细胞;降低p21基因表达并抑制氧化损伤后晶状体上皮细胞凋亡可能是其保护机制之一。     

Effects of calcium on lens epithelial cells in rabbits

PURPOSE: The action of lens epithelial cells (LECs) is important for cataract and posterior subcapsular cataract after cataract surgery. In this study, we analyzed the effects of calcium on the characteristics of LECs. METHODS: The LECs were collected using albino rabbits and incubated in minimum essential medium [MEM, Introgen Corp. (12% fetal bovine serum: FBS)] (37 degrees C, 5 % CO2) for a week to induce their proliferation. Cell culture dishes (35 mm) were prepared and 7 mm cylindrical pipes were placed in them. After that, around 10,000 cultured LECs were placed in the pipes and incubated. After 2 hours incubation, the pipes were removed and various doses of MEM (0, 2, 10 and 20 mM) replaced the calcium. Proliferation and shapes of LECs were observed using a confocal microscope and immunohistological analysis [alpha-smooth muscle actin (alpha-SMA) and bromodeoxyuridine (BrdU)]. The LECs were incubated with collagen gel and different calcium doses (0, 2, 10 and 20 mM) of MEM to calculate the contraction rate. RESULTS: It was observed that the LECs changed to fibroblast-like cells at high doses of calcium using a confocal microscope. Histological studies showed that the BrdU positive cells were increased by using 10 and 20 mM calcium MEM, but the positive cells were decreased by using 0 and 2 mM calcium MEM. Increase of alpha-SMA stained cells was recognized when using 0, 10 and 20 mM calcium MEM. The contraction rate of collagen gel was increased by using the 10 and 20 mM calcium MEM. CONCLUSION: The changes of calcium concentration might be an important factor for the development of cataract, posterior subcapsular opacification, and contraction of the lens capsule after cataract surgery.     

过氧化氢对体外培养的视网膜色素上皮细胞增殖及DNA合成的影响

目的 探讨过氧化氢(hydrogen peroxide,H_2O_2)对体外培养的人胚胎视网膜色素上皮(RPE)细胞的抑制作用及DNA合成的影响。方法 分别用MTT法测定加入0、50、100、200、400、500μmol/L的H_2O_2作用60h后RPE细胞数量的改变和核酸蛋白分析仪测定加入50、100、200、300、400、500μmol/L H_2O_2作用60h后RPE细胞NDA浓度的变化。结果50、100、200、400、500μmol/L的H_2O_2作用60h后其细胞A值分别为(0.1731±0.0260)、(0.1590±0.0418)、(0.1491±0.0265)、(0.1506±0.0224)、(0.1538±0.0221)与对照组(0.1978±0.0223)相比,差异有显著性(/值分别为1.961、3.082、3.868、3.749、4.932,P值分别为0.054、0.003、0.000、0.000、0.000);50、100、200、300、400、500μmol/L H_2O_2作用60h后其细胞DNA浓度分别为(247.5225±36.8007)、(138.3373±14.6158)、(1043.3095±14.2524)、(85.6586±14.8986)、(70.2618±11.8879)、(56.1955±12.8999)μmol/L,与对照组(293.9311±51.7510)μmol/L相比,差异有显著性(t值分别为3.659、12.376、15.083、16.566、17.791、18.909,P值分别为0.001、0.000、0.000、0.000、0.000、0.000)。结论H_2O_2能抑制体外培养的RPE细胞增殖及DNA合成。     

曲尼司特对培养兔晶状体上皮细胞的作用

目的 观察曲尼司特(Tranilast)对培养兔晶状体上皮细胞增殖的作用及其对细胞周期的影响。方法 将传代的兔晶状体上皮细胞用含不同曲尼司特浓度的培养液培养，每天计数，绘制细胞生长曲线，收集细胞做流式细胞仪检查。空白培养液培养的细胞做阴性对照，含0．004％丝裂霉素C(MMC)培养液培养的细胞为阳性对照。结果 用180μmol／L的培养液培养细胞时细胞增殖明显受到抑制，随药物浓度增加，细胞增殖受抑制越明显，细胞生长曲线越低平，细胞形态变化不大。经流式细胞术进行细胞周期分析，随药物浓度增加G0／G1期细胞数增多(67．47％～79％)，而S期细胞数减少(21．19％～4．32％)。阳性对照组细胞于48h全部死亡。结论 曲尼司特具有抑制晶状体上皮细胞增殖的效果，且呈浓度依赖性。曲尼司特将细胞抑制在G1期的限制点内。     

Activity of glutathione peroxidase and glutathione reductase in the human lens related to age

Lenses from 42 eye bank eyes were assayed for glutathione peroxidase and glutathione reductase activities. The activity of glutathione peroxidase, when considered as a function of age, was lowest in the neonate lens, increasing with age to reach maximal values in young adult lenses, and thereafter progressively decreasing with ages greater than 40 years. Glutathione reductase activity was little affected by age when expressed as activity per lens, per gram lens or per mg soluble protein, indicating that activity of this enzyme did not increase with lens size as would a representative lenticular protein. However, the activity of this enzyme per gram lens was among the highest of any species yet examined.     

Reversal of galactosemic-induced inhibition of PGH synthase activity in cultured lens epithelial cells

Inhibition of prostaglandin synthesis as determined by prostaglandin endoperoxide synthetase (PGH synthase) activity is associated with polyol accumulation in cultured bovine lens epithelial cells (BLECs) incubated six days in minimal essential medium (MEM) containing 40 mM galactose (Gal). In order to better understand the nature of the correlation between hypergalactosemic exposure, polyol accumulation and inhibition of prostaglandin synthesis, a series of culture media reversal and sorbinil (an aldose reductase inhibitor) addition studies were carried out. BLECs were incubated in Gal for six days, then changed to galactose-free MEM +/- sorbinil for a three day recovery period. PGH synthase activity reduced to 66% of control after six days of exposure to Gal. The simultaneous administration of sorbinil during a nine day Gal incubation significantly protected the enzymatic activity, while the activity of PGH synthase further declined to 41% of control under the same conditions in the absence of sorbinil. Within 72 hours of media reversal, PGH synthase activity equaled or exceeded control values in BLECs switched to either MEM or MEM + sorbinil. Indeed, an enhanced prostaglandin biosynthetic capacity as demonstrated by radioimmunoassay was exhibited with microsomes prepared from cells switched from Gal into Gal-free MEM +/- sorbinil, corroborating the beneficial effect of media reversal. Furthermore, following 72 hours of reversal, the cellular dulcitol level was 93 nmol/micrograms PO4 for BLECs switched to MEM alone; no detectable level of polyol was observed in BLECs changed to MEM + sorbinil. In contrast, the polyol content in BLECs after six days of exposure to Gal was 185 nmol/micrograms PO4 and increased to 334 nmol/micrograms PO4 after nine days of continuous incubation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of cycloheximide on Na,K-ATPase activity in cultured human lens epithelial cells

PURPOSE: Earlier studies from this laboratory demonstrated the ability of lens epithelium to synthesize new Na,K-adenosine triphosphatase (Na,K-ATPase) catalytic subunit (alpha) polypeptide under conditions of increased ion permeability. In the present study, the authors considered whether continuous synthesis of Na,K-ATPase protein is necessary for maintenance of Na,K-ATPase activity in lens cells. METHODS: Na,K-ATPase activity was measured by quantifying the ouabain-sensitive rate of ATP hydrolysis in cultured human lens epithelial cells (HLE-B3) permeabilized with digitonin. The abundance of Na,K-ATPase alpha subunit was determined by Western blot analysis. Synthesis of Na,K-ATPase alpha1 polypeptide was investigated by measuring 35S-methionine incorporation. RESULTS: Na,K-ATPase activity was reduced to less than 20% of the control level in HLE-B3 cells exposed to 100 microM cycloheximide for 24 hours. However, as judged by Western blot density, the abundance of Na,K-ATPase alpha1 and alpha3 subunit in cycloheximide-treated cells was 90% and 84% of the control level, respectively. 35S-methionine incorporation experiments revealed detectable labeling of Na,K-ATPase alpha1 subunit polypeptide within 30 minutes, consistent with alpha1 polypeptide synthesis. Na,K-ATPase alpha1 polypeptide labeling was also detected in the epithelium of intact rat lenses that had been allowed to incorporate 35S-methionine. Cycloheximide abolished 35S-methionine incorporation into Na,K-ATPase alpha1 subunit polypeptide of HLE-B3 cells. When added during the chase phase of the experiment, cycloheximide was found to slow the disappearance of labeled alpha1 polypeptide, consistent with a reduced rate of polypeptide degradation. CONCLUSIONS: The results suggest that a continuous cycle of Na,K-ATPase alpha1 synthesis and degradation may occur in lens epithelial cells. Cycloheximide appeared to inhibit Na,K-ATPase protein synthesis and degradation. The observed reduction of Na,K-ATPase activity after treatment with cycloheximide indicates that even though Na,K-ATPase remains abundant, Na,K-ATPase becomes inactivated when protein synthesis is inhibited.     

PDTC对过氧化氢诱导鼠晶状体上皮细胞NF-κB活化表达的影响

目的　观察过氧化氢 (H2 O2 )诱导鼠白内障形成过程中晶状体上皮细胞核因子 KB(NF κB)的活化表达及吡咯烷二硫氨基甲酸 (PDTC)对核因子κB(NF κB)活化表达的抑制作用 ,探讨NF κB及PDTC在白内障发生、预防和治疗中的作用。方法　大鼠晶状体器官离体培养 ,免疫组织化学方法检测晶状体上皮细胞NF κB的活化表达。结果　随过氧化氢损伤时间的延长 ,H2 O2 组晶状体上皮细胞NF κB活化表达逐渐增强 ,且与晶状体混浊程度呈正相关。PDTC可以明显抑制晶状体上皮细胞NF κB阳性活化表达和减轻晶状体混浊程度 ,且存在一定程度的剂量依赖性。结论　过氧化氢可以诱导鼠晶状体上皮细胞NF κB的活化表达 ,PDTC可以有效抑制NF κB的活化表达 ,有可能对抗或延缓白内障的发生     

Phosphorus-31 NMR study of the effects of hydrogen peroxide on young and old rat lenses

31P-NMR spectroscopy was used to study the dynamic changes in young and old rat lenses under oxidative stress with hydrogen peroxide. Control spectra were recorded for young and old rat lenses using normal media. Oxidative stress spectra were recorded under the same conditions, except that the normal media also contained hydrogen peroxide at four different concentrations. With increasing H2O2 concentration in the perfusion media there was a corresponding decrease in the observed phosphorus metabolites, phosphorylcholine and ATP. There was significant difference in the rate of depletion of metabolites between the young and old rat lenses; old rat lens showed an ATP decrease almost double that for young rat lenses. Also, the 31P control spectrum of the old rat lens was different from that of the young lens. The NMR results showed the importance of comparison of old and young rat lenses under oxidative stress as a model for senile cataractogenesis.     

Detoxification of H2O2 by cultured rabbit lens epithelial cells: participation of the glutathione redox cycle

Although it has been shown that cultured rabbit lenses can adequately defend against the 0.03-0.05 mM level of H2O2 normally found in aqueous humor, the contribution of the epithelium in this process has not been well defined. In the present study, the peroxide-detoxifying ability of the epithelium is evaluated in cultured rabbit lens cells established from 4-6-day-old rabbits and compared to that of skin fibroblasts from rabbits of the same age. When cells were cultured in medium containing H2O2, the concentration of peroxide rapidly decreased; however, various concentrations could be maintained for 3-hr periods by using glucose oxidase to enzymically generate H2O2. At an extracellular level of 0.03 mM H2O2, the rate of detoxification of peroxide by epithelial cells was 2 mumol H2O2 (8 x 10(5) cells)-1 3 hr-1, twice as fast as that for fibroblasts. Epithelial cells contained a high level of reduced glutathione (GSH) equal to 36 nmol (8 x 10(5) cells)-1, twice that present in the fibroblasts. The concentration of GSH in 8 x 10(5) epithelial cells, a number of cells normally present in one intact rabbit lens epithelium, remained constant during 3 hr of exposure to H2O2 levels as high as 0.03 mM, even though the amount of H2O2 taken up under these conditions was sufficient to oxidize completely the cellular GSH every 2 min. In contrast, the GSH content of fibroblasts declined at levels of peroxide above 0.01 mM. Participation of the glutathione redox cycle in the H2O2-detoxification process was demonstrated from studies of hexose monophosphate shunt (HMPS) activity as measured by oxidation of [1-14C]-labeled glucose. The oxidation of [1-14C]-glucose in epithelial cells was stimulated 13 times that of controls during exposure to 0.04-0.05 mM H2O2, while the corresponding increase in oxidation of [6-14C]-labeled glucose was only 1.6 times. In contrast, maximum shunt activity in fibroblasts occurred at 0.03-0.04 mM H2O2 and was six times the control value. The growth potential of the cells following a 3-hr exposure to H2O2 was also used as a measure of oxidant toxicity in both cell types. Concentrations of H2O2 up to 0.03 mM had no effect on the growth of 8 x 10(5) epithelial cells but did diminish the growth of the same number of fibroblasts. Cell density was found to be an important parameter in the ability of the cells to tolerate H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)     

小牛晶状体上皮细胞传代培养的细胞学特征

目的通过建立小牛晶状体上皮细胞的传代培养,观察晶状体上皮细胞的离休生长特性。方法组织块接种培养;用计数法及ＭＴＴ法测细胞生长曲线;绘制细胞分裂指数曲线。结果采用组织块培养法晶状体上皮细胞原代及传代培养生长良好;ＭＴＴ及细胞计数法所测细胞生长过程基本一致;细胞分裂指数曲线显示细胞在培养第５天达分裂高峰。结论组织块法是晶状体上皮细胞离体培养的较好方法;晶状体上皮细胞具有较好的离体生长能力;ＭＴＴ比色法是检测晶状体上皮细胞的有效方法。