密闭囊袋冲洗系统模拟抑制人晶状体上皮细胞的研究

目的 利用密闭囊袋冲洗系统和人后发性白内障(PCO)囊袋模型,研究三氧化二砷(As2O3)在短时间内对PCO的防治作用.方法 实验研究.严格将时间控制在2 min时,在细胞培养条件下,四甲基偶氮唑盐比色法观察As2O3对人晶状体上皮细胞(LEC)系FHL124细胞的抑制作用;乳酸脱氢酶释放实验观察As2O3对FHL124细胞的损伤作用;荧光显微镜动态检测细胞内Ca2+浓度的变化.建立人PCO囊袋模型,应用密闭囊袋冲洗系统观察As2O3对原代人LEC的作用.浓度反应曲线用Hill公式求IC50值.所有实验数据结果用均数±标准差表示,采用SPSS 13.0软件处理,组间差异用单因素方差分析,组间两两比较用Dunnett检验.结果 As2O3在作用仅2 min的情况下,就可以抑制人LEC和清除残留于囊袋内的人原代LEC.As2O3(10、30、100、300、1000 μmol/L)对FHL124细胞的作用呈剂量依赖性,100 μmol/L以上浓度的As2O3对FHL124细胞具有明显的毒性作用.与对照组比较,差异有统计学意义(t=5.217,P＜0.01),半效抑制量(IC50)=130 μmol/L.乳酸脱氢酶检测显示,As2O3可影响细胞膜结构的完整性.细胞内动态Ca2+检测显示,As2O3对细胞内Ca2+作用呈剂量依赖性.高浓度As203可引起细胞内Ca2+库释放,同时抑制细胞的Ca2+内流,最终导致细胞内Ca2+稳态的破坏而引起细胞的死亡.1×104 μmol/L As2O3作用2 min后,使Ca2+内流的幅值及速度分别降低了(43.24±2.98)%(t=3.134,P＜0.01)和(46.27±6.01)%(t=3.521,P＜0.01).人PCO囊袋模型显示,联合应用As2O3和密闭囊袋冲洗系统可以在2 min内清除残留于囊袋内的原代人LEC.结论 As2O3可以在短时间内彻底清除白内障手术中存留于囊袋内的人LEC,与密闭囊袋冲洗系统结合可能抑制PCO的发生.     

三氧化二砷对表皮生长因子诱导的视网膜色素上皮细胞增生和迁移的影响

背景 细胞因子失衡所导致的视网膜色素上皮(RPE)细胞的异常增生和迁移是增生性玻璃体视网膜病变( PVR)的主要病理变化之一.三氧化二砷(As2O3)是中国传统中药中的有效成分,可有效抑制肿瘤细胞的增生和迁移.但As2O3对细胞生长因子引起的RPE细胞增生和迁移的影响尚未明确. 目的 探讨As2O3对表皮生长因子(EGF)诱导的ARPE-19细胞增生和迁移的影响.方法 用无血清培养基对RPE细胞系ARPE-19细胞进行培养,将终浓度为0、0.5、1.0、2.0、5.0、10.0和20.0 μmol/L的As2O3分别加入到无血清培养基和含10 mg/L EGF的ARPE-19细胞培养液中作用24 h和48 h,通过噻唑蓝(MTT)比色法检测各培养组ARPE-19细胞活性的吸光度(A)值,以探讨As2O3对细胞的药物毒性作用,并筛选安全、有效的As2O3作用浓度.用10 mg/L EGF加入培养基诱导ARPE-19细胞迁移,分别在培养板中加入0、0.5、1.0、2.0μmol/L As2O3 作用24 h和48 h,并通过划痕试验和Transwell试验检测As2O3对EGF诱导的ARPE-19细胞迁移的影响.结果 MTT法检测发现不同浓度As2O3组作用24 h和48 h后,无血清培养组细胞A值随As2O3浓度的升高而逐渐下降,总体差异有统计学意义(F浓度=38.269,P=0.000;F时间=0.874,P=0.358).与空白对照组(0μmol/LAs2O3组)比较,0.5～ 5.0 μmol/L As2O3组ARPE-19细胞A值的差异均无统计学意义(P＞0.05).对含10 mg/LEGF组的ARPE-19细胞,药物对细胞A值的影响呈现浓度和时间依赖性(F浓度=152.155,P=0.000;F时间=51.649,P=0.000).与对照组比较,0.5～2.0μmol/L As2O3加入24 h和48 h后,A值的变化差异均无统计学意义(P＞0.05),而0.5、1.0、2.0μmol/L As2O3加入10 mg/L EGF诱导的ARPE-19细胞中作用24h和48 h后,A值的变化差异均无统计学意义(F浓度=2.215,P=0.126;F时间 =2.230,P=0.155).5.0 ～20.0 μmol/L As2O3作用于EGF诱导的ARPE-19细胞中作用后,细胞A值明显下降,与空白对照组比较差异均有统计学意义(P＜0.05),5.0～20.0 μmol/L As2O3作用24 h后,对EGF诱导的ARPE-19细胞增生抑制率分别为12％、32％、37％;作用48 h后细胞抑制率分别为39％、44％和53％.划痕试验结果显示,0.5～2.0 μmol/L As2O3对EGF诱导的ARPE-19细胞的横向迁移具有抑制作用.Transwell试验结果表明,0.5～2.0 μmol/L As2O3对10 mg/L EGF诱导的ARPE-19细胞纵向迁移有明显的抑制作用,0.5、1.0、2.0μmol/L As2O3作用12h对ARPE-19细胞的抑制率分别为22％、33％和46％. 结论 As2O3在一定浓度范围内对ARPE-19细胞无毒性作用,2.0 μmol/L以下浓度的As2O3对EGF诱导的ARPE-19细胞增生无明显影响,但可影响细胞的迁移能力,5.0 μmol/L以上浓度的As2O3可明显抑制ARPE-19细胞的增生.     

△ψm和Caspase3在As_2O_3诱导腺样囊性癌ACC-2细胞凋亡过程中的作用

目的：探讨线粒体膜电位（△ψm）、Caspase3在As2O3诱导ACC-2细胞凋亡中的作用。方法：进行ACC-2细胞培养,将As2O3建立不同药物浓度梯度（0,1.0,2.0,4.0,8.0μmol/L）分别作用于ACC-2细胞,用Rh123染色,流式细胞仪检测8.0μmol/LAs2O3作用前、后（24h）,ACC-2细胞的线粒体膜电位（△ψm）变化;用多功能酶标仪进行Caspase3活性检测。结果：空白对照组ACC-2细胞内Rh123荧光强度最强,8.0μmol/LAs2O3处理组ACC-2细胞内Rh123荧光强度减弱,其差异有显著性（P〈0.05）;随着As2O3药物浓度的增高（0,1,2,4,8μmol/L）,ACC-2细胞的Caspase3酶活力单位逐渐增加。结论：As2O3作用于ACC-2细胞,可通过降低线粒体膜电位从而引起细胞凋亡。随着As2O3药物浓度的增高,ACC-2细胞的Caspase3酶活力单位逐渐增加,Caspase3被激活,细胞可发生不可逆转的凋亡过程。     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

目的 探讨褪黑素(Mel)对过氧化氢(H202)氧化损伤的人视网膜色素上皮(hRPE)细胞的保护作用及其作用机制.方法 实验研究.采用600μmoL/L H2O2建立体外培养的hRPE细胞氧化损伤模型.实验分为6组:溶剂对照组、600μmoL/L H2O2+溶剂组(H2O2损伤模型组)、600μmoL/L H2O2+10-7mol/L Mel组、600μmoL/L H2O2+10-6moL/L Mel组、600 μmol/L H2O2+10-5mol/L Mel组、600μmol/L H2O2+10-4moL/L Mel组.通过四甲基偶氮唑盐(MTT))法检测细胞活性;测定细胞内超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量以反映细胞氧化损伤程度;分别用DNA Ladders电泳法和流式细胞仪检测细胞的凋亡情况.溶剂对照组与600μmol/L H2O2组间均数比较采用随机区组设计的t检验;600μmol/L H2O2组以及600μmol/L H2O2+不同浓度Mel组间均数比较采用单因素5水平设计的方差分析,组间两两比较采用LSD-t检验.结果 H2O2模型组较对照组细胞活性明显降低、SOD活件降低、MDA含量增加、凋亡率升高,差异均有统计学意义(t=2.25,39.50,68.42;P<0.05);Mel干预组较模型组细胞活性升高、SOD活性升高、MDA含最减少、凋亡率降低,差异有统计学意义(P<0.05),并与药物浓度的变化呈正相关趋势.结论 Mel对H2O2诱导的RPE的氧化损伤具有保护作用,其机制可能与影响细胞活性、增强抗氧化酶活性、减少细胞凋亡有关.     

Src-家族酪氨酸激酶特异性抑制剂对H2O2介导的晶状体上皮细胞Ca2＋内流的影响

目的观察Src-家族酪氨酸激酶（SFK）抑制剂PP1对H2O2诱导的晶状体上皮细胞（LECs）内游离钙离子（Ca2＋）浓度变化的影响。方法用Ca2＋荧光探针Fluo-3/AM负载人晶状体HLE-B3,用激光共焦显微镜观察在不同浓度H2O2刺激后细胞内Ca2＋的荧光强度变化;进一步用0.1nmol/LPP1、0.3μmol/（L·min）过氧化氢酶和DMSO分别预处理细胞,观察在常规Ca2＋、低Ca2＋和高Ca2＋培养基条件下H2O2刺激后细胞内Ca2＋荧光强度的变化,评价PP1对H2O2诱导的LECs内Ca2＋的作用。结果不同浓度H2O2刺激后细胞内游离Ca2＋浓度升高,呈剂量依赖效应。用0.1mmol/LH2O2刺激细胞,在常规Ca2＋培养基中,PP1组和过氧化氢酶组细胞内Ca2＋荧光强度增长幅度与DMSO组比较分别降低（28.5±4.2）%、（33.8±3.7）%,差异均有统计学意义（q=3.73,P〈0.05;q=4.21,P〈0.05）。在低Ca2＋培养基中,3个组细胞内Ca2＋荧光强度增强均不明显;在高Ca2＋培养基中,PP1组、过氧化氢酶组的荧光强度增加幅度较DMSO组分别降低（13.5±1.8）%和（21.3±2.4）%（q=5.58,P〈0.01;q=7.11P〈0.01）。常规Ca2＋和高Ca2＋培养基中,PP1组和过氧化氢酶组细胞内Ca2＋荧光强度增长幅度的差异均无统计学意义（q=3.04,P〉0.05;q=2.76,P〉0.05）。结论 SFK特异性抑制剂PP1能有效抑制H2O2诱导的LECs的Ca2＋内流,从而阻断依赖Ca2＋激活的信号转导途径,阻止皮质性白内障的发生。     

虾青素对晶状体上皮细胞氧化应激损伤的抑制作用及其机制

目的研究虾青素对过氧化氢(H2O2)诱导晶状体上皮细胞HLEB-3氧化应激损伤的调控作用及其可能的作用机制。方法将HLEB-3细胞于不同浓度H2O2(0、50、100、200、500、750 μmol/L)下培养, 噻唑蓝(MTT)法检测细胞抑制率, 计算半数抑制浓度(IC50)。将HLEB-3细胞使用不同浓度(0、5、10、20、50 μmol/L)虾青素培养, MTT法检测细胞存活率。将HLEB-3细胞分为4个组, 其中正常对照组用完全培养基培养、氧化应激组于250 μmol/L H2O2培养基中培养, 10 μmol/L虾青素组和20 μmol/L虾青素组分别于相应浓度虾青素+250 μmol/L H2O2培养基中培养, 各组均培养24 h。采用流式细胞仪检测细胞凋亡率;采用ELISA法检测细胞一氧化氮(NO)浓度、超氧化物歧化酶(SOD)活性、还原型谷胱甘肽(GSH)活性和丙二醛(MDA)含量;采用Western bolt法检测细胞核核因子E2相关因子2(Nrf2)、细胞质Nrf2和血红素加氧酶-1(HO-1)、醌氧化还原酶1(NQO1)蛋白表达。另将细胞分为正常对照-小干扰R...     

神经生长因子对N-甲基-D-门冬氨酸诱导人胚胎RPE细胞凋亡的保护作用

目的 探讨神经生长因子(NGF)对体外培养的人胚胎视网膜色素上皮(hFRPE)细胞凋亡的保护作用。方法 将传代培养的hFRPE细胞,用200μmol/L,300μmol/L N-甲基-D-门冬氨酸(NMDA)建立诱导的hFRPE细胞凋亡模型,并用吖啶橙(AO)荧光染色计数和透射电镜(TEM)观察NGF对凋亡细胞的保护作用。 结果 用AO荧光染色及TEM均观察到经200 μmol/L,300μmol/L NMDA处理72 h后的hFRPE细胞出现典型的凋亡形态学改变。200μmol/L.NMDA作用72 h后hFRPE细胞凋亡指数为(32.587 9±1.488 0)％和200 μmol/L NMDA+300 μmg/L NGF作用72 h后的hFRPE细胞凋亡指数(20.768 7±1.302 4)％相比,差异有显著性(q=2.941 4,P=0.005 0);300 μmol/L NMDA作用72 h后hFRPE细胞凋亡指数为(42.506 4±3.014 8)％,与300 μmol/L NMDA+300 μg/L NGF作用72 h后的hFRPE细胞凋亡指数(25.687 2±1.8974)％相比,差异有非常显著性的意义(q=4.117 8,P=0.000 0)。 结论 NCF能拈抗NMDA诱导的hFRPE细胞凋亡。     

应用反义寡核苷酸沉默PDGFR-α表达对RPE细胞增生和凋亡的影响

背景 视网膜色素上皮( RPE)细胞能分泌血小板源性生长因子(PDGF),又具有PDGF受体(PDGFR),已有研究表明PDGF对增生性玻璃体视网膜病变(PVR)的形成起着关键性作用. 目的 应用反义寡核苷酸( ASODN)技术抑制血小板源性生长因子受体-α(PDGFR-α)基因的表达,观察其对RPE细胞增生和凋亡的影响.方法 将人RPE细胞株在含质量分数10％胎牛血清的低糖DMEM培养基中进行培养,取对数生长期细胞调节细胞密度为5×105个/孔,接种于96孔板,待细胞生长至80％～90％融合时进行转染.空白对照组不加PDGFR-α ASODN及阳离子脂质体Lipofectamine 2000,1.0μmol/L Lipo-ASODN组、2.0μmol/LLipo-ASODN组使用阳离子脂质体Lipofectamine 2000将靶向PDGFR-α基因的ASODN转染至RPE细胞株.细胞转染48 h后,MTT法检测各组细胞的吸光度(A)值并以A490表示,逆转录聚合酶链反应(RT-PCR)法检测PDGFR-α mRNA在RPE细胞中表达的变化;hoechst 33258荧光染色观察培养细胞的凋亡情况;流式细胞仪检测RPE细胞的细胞周期和凋亡率. 结果 空白对照组、1.0 μmol/L Lipo-ASODN组及2.0μmol/L Lipo-ASODN组RPE细胞的A490值分别为1.45±0.12、1.07±0.06、0.65±0.05,差异有统计学意义(F=97.72,P=0.00),1.0μmol/L Lipo-ASODN组和2.0 μmol/L Lipo-ASODN组RPE细胞A490值明显低于空白对照组,差异均有统计学意义( P=0.00、0.00);2.0 μmol/L Lipo-ASODN组RPE细胞A490值明显低于1.0 μmol/L Lipo-ASODN组,差异有统计学意义(P=0.00).Hoechst 33258荧光染色显示,转染PDGFR-α ASODN后RPE细胞凋亡数量多于空白对照组.流式细胞仪检测表明,转染PDGFR-α ASODN后RPE细胞阻滞于G0/G1期(F=206.70,P=0.00),1.0 μmol/L Lipo-ASODN组和2.0μmol/L Lipo-ASODN组RPE细胞凋亡率均明显高于空白对照组(37.8±1.3 vs 10.5±0.1,61.2±1.9 vs 10.5±0.1)(F=1808.90,P=0.00).PDGFR-α在RPE细胞中的表达随PDGFR-α ASODN浓度的升高有降低的趋势. 结论 利用ASODN技术沉默PDGFR-α基因表达可以显著抑制PVR过程中RPE细胞的增生,并能诱导其凋亡,不同浓度PDGFR-α ASODN转染后能下调PDGFR-α mRNA的表达,PDGFR-α基因的ASODN靶向技术为PVR的基因治疗提供了实验依据.     

不同浓度维拉帕米及不同光照形式对豚鼠RPE细胞Ca2+的影响

目的:探讨不同浓度维拉帕米(verapamil,Ver)对体外培养豚鼠视网膜色素上皮(retinal pigment epithelium,RPE)细胞内Ca2+浓度的影响,并比较有无Ver作用下经不同形式光照后RPE细胞内Ca2+浓度的变化。方法:2周龄幼年健康豚鼠10只,体外培养RPE细胞,传代、鉴定后,将细胞分为Ver处理组和未处理组,Ver处理组加入80mg/LVer作用12h。两组均进一步分为聚焦光组、离焦光组、平行光组和空白对照组,前三组分别接受聚焦光、离焦光(均为将平行光经透镜转化)和平行光照射,空白对照组不接受照射。于照射后立即采用激光扫描共焦显微镜(laser scanning confocal microscopy,LSCM)测定细胞内Ca2+荧光强度,分析不同光照形式与效应的关系。统计学方法采用单因素方差分析。结果:Ver作用于RPE细胞12h后,20,40,80mg/L组的细胞凋亡情况与空白对照组相比均无统计学意义(P>0.05),Ver可降低RPE细胞内Ca2+荧光强度,浓度为20,40,80mg/L时分别降低了10.36%,24.54%,58.05%,仅80mg/L组与无光照组相比差异有统计学意义(P<0.05)。未加Ver对RPE细胞进行光照,聚焦光组的Ca2+荧光强度较其他各组明显升高,离焦光组的荧光强度也较高,组间比较有统计学差异(P<0.05)。加入80mg/LVer对RPE细胞作用12h后再进行光照,光照各组Ca2+荧光强度没有明显提高,组间比较没有统计学差异(P>0.05)。结论:超过一定浓度的Ver可诱导RPE细胞凋亡,80mg/L可在不引起RPE细胞凋亡的前提下有效降低细胞内Ca2+荧光强度;不同光照形式对豚鼠RPE细胞内Ca2+有明显刺激作用,聚焦光影响最大;不同光照形式对Ver处理的RPE细胞内的Ca2+荧光强度无明显作用。     

维拉帕米诱导人视网膜色素上皮细胞凋亡及细胞内钙变化

目的:探讨钙通道拮抗剂—维拉帕米(verapamil,Ver)对体外培养的人视网膜色素上皮(humanretinalpigmentepithelium,hRPE)细胞凋亡诱导作用及凋亡过程中细胞内钙浓度[Ca2 ]i的变化。方法:应用80mg/L的Ver作用体外培养hRPE细胞12,24及48h,采用吖叮橙(AO)荧光染色法、透射电镜、流式细胞术观察hRPE细胞凋亡;Fluo-3/AM负载技术,观察凋亡过程中细胞[Ca2 ]i的变化。结果:一定浓度Ver可诱导hRPE细胞凋亡,荧光显微镜及电镜观察hRPE细胞具有早期凋亡特征:核荧光呈黄绿色,电镜下核染色质浓染、边集。流式细胞术显示,Ver作用后的hRPE细胞凋亡百分率增加(F=12.3415,P<0.05)。Ver作用的hRPE细胞内钙浓度([Ca2 ]i)呈下降趋势(F=23.607,P<0.01)。结论Ver能诱导体外培养的hRPE细胞凋亡,细胞内[Ca2 ]i浓度的变化可能是诱导hRPE凋亡的机制之一。     

Arsenic trioxide initiates ER stress responses, perturbs calcium signalling and promotes apoptosis in human lens epithelial cells

Identification of novel agents to eradicate the residual lens cell population following cataract surgery provides one mode of preventing PCO formation. The present study investigated the biological mechanism of As(2)O(3) cytotoxicity in a human lens cell line and capsular bag system. FHL 124 cell survival was assessed by quantification of total protein content, a cell population measure. Gene changes were detected by Real-time PCR; apoptosis by TUNEL assays. Intracellular calcium was measured by real-time fluorimetric single-cell digital imaging techniques after Fura-2 incorporation. In vitro human capsular bags were generated from donor eyes, which involved sham cataract surgery then use of the Perfect Capsule device to form a closed system to deliver As(2)O(3) for 2min. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence immunocytochemistry. FHL 124 cells demonstrated a dose-dependent sensitivity to As(2)O(3) exposure. A 2min exposure of As(2)O(3) to cells within the capsular bag, using the perfect capsule system, resulted in total cell death when used at 100mM. As(2)O(3) provoked an ER stress response identified through an upregulation of known genes. As(2)O(3) depleted the calcium store and consequently lead to reduced calcium signalling. As(2)O(3) increased rates of apoptosis. Arsenic trioxide provokes ER stress that leads to down-regulation of calcium signalling resulting in apoptosis. The application of As(2)O(3) to cells within the capsular bag for a 2min window using the Perfect Capsule system predicts putative therapeutic benefit in vivo.     

Growth factor receptor signalling in human lens cells: role of the calcium store

In the human lens, stimulation of tyrosine-kinase coupled growth factor receptors such as epidermal growth factor receptor (EGFR) can induce calcium release from endoplasmic reticulum (ER) stores. The present study investigated the impact of calcium store inactivation on EGFR signalling, cell growth and death in a well-characterised human lens cell line (FHL124). FHL124 cells were routinely cultured in Eagle's minimum essential medium (EMEM) supplemented with 10% foetal calf serum (FCS) and seeded on 24-well plates (DNA and protein synthesis), tissue culture dishes (growth assay, western immunoblot), and glass coverslips (immunocytochemistry). DNA and protein synthesis rates were quantified by measuring the incorporation of (3)H-thymidine and (35)S-methionine into FHL124 cells in serum-free EMEM or EMEM supplemented with thapsigargin (Tg) (100 nM and 1 microM). Longer-term growth was assessed by quantifying the increase in area over time of a circular patch of seeded cells. EGFR was identified using anti-EGFR mouse monoclonal antibody and visualised by fluorescence microscopy with ALEXA 488 conjugated secondary antibody. Programmed cell death was determined by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay method. Activation of the mitogen-activated protein kinase (MAPK) signalling protein extracellular signal-regulated kinase (ERK) and the cell cycle proteins CDK2 and P27(kip1) were detected by western immunoblot techniques. Inactivation by > or =100 nM Tg inhibited both protein and DNA synthesis although the effect on the latter was greatest. The cell cycle activator CDK2 was reduced by Tg, while the inhibitor P27(kip1) was increased along with the percentage of apoptotic cells. A single, maximal epidermal growth factor (EGF) (10 ng ml(-1)) exposure induced receptor internalization and increased ERK phosphorylation. Both internalisation and ERK activation were unaffected by the presence of Tg. However, reduced internalisation and ERK activation followed repeated EGF applications in the presence of Tg. Additionally, ERK activation by submaximal EGF concentrations was reduced by store depletion. An intact endoplasmic reticulum calcium store therefore plays a significant role in human lens cell survival and growth.     

Arsenic trioxide initiates ER stress responses,perturbs calcium signalling and promotes apoptosis in human lens epithelial cells

Identification of novel agents to eradicate the residual lens cell population following cataract surgery provides one mode of preventing PCO formation. The present study investigated the biological mechanism of As₂O₃ cytotoxicity in a human lens cell line and capsular bag system. FHL 124 cell survival was assessed by quantification of total protein content, a cell population measure. Gene changes were detected by Real-time PCR; apoptosis by TUNEL assays. Intracellular calcium was measured by real-time fluorimetric single-cell digital imaging techniques after Fura-2 incorporation. In vitro human capsular bags were generated from donor eyes, which involved sham cataract surgery then use of the Perfect Capsule device to form a closed system to deliver As₂O₃ for 2 min. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence immunocytochemistry. FHL 124 cells demonstrated a dose-dependent sensitivity to As₂O₃ exposure. A 2 min exposure of As₂O₃ to cells within the capsular bag, using the perfect capsule system, resulted in total cell death when used at 100 mM. As₂O₃ provoked an ER stress response identified through an upregulation of known genes. As₂O₃ depleted the calcium store and consequently lead to reduced calcium signalling. As₂O₃ increased rates of apoptosis. Arsenic trioxide provokes ER stress that leads to down-regulation of calcium signalling resulting in apoptosis. The application of As₂O₃ to cells within the capsular bag for a 2 min window using the Perfect Capsule system predicts putative therapeutic benefit in vivo.     

Hepatocyte growth factor function and c-Met expression in human lens epithelial cells

PURPOSE: Hepatocyte growth factor (HGF) and its receptor c-met perform a multitude of functions. However, despite the significant degree of study of HGF and c-met in numerous tissues and cell types, relatively few investigations have been performed on the lens. In the current study, therefore, the role of HGF and the receptor c-met in human lens epithelial cells was investigated. METHODS: Anterior epithelium and capsular bags were prepared from human donor eyes and maintained in Eagle's minimum essential medium (EMEM) in a 5% CO(2) atmosphere at 35 degrees C. In addition, the human lens cell line FHL124, was routinely cultured and seeded onto glass coverslips (c-met immunodetection), 12-well plates (DNA and protein synthesis), and tissue culture dishes (migration). c-Met was detected by immunocytochemistry and fluorescence-activated cell scanning (FACS). HGF was measured using enzyme-linked immunosorbent assay (ELISA) techniques. Proliferation and protein synthesis were determined by [(3)H]thymidine and (35)S-methionine incorporation into DNA and proteins, respectively. Migration was assessed using a scratch-wound assay and time-lapse video microscopy. RESULTS: HGF was detected at all stages of culture of capsular bags in protein-free medium. Moreover, c-met was present on the native epithelium and after mechanical trauma was seen to be upregulated. Immunolocalization and FACS analysis demonstrated c-met expression on FHL124 cells throughout the whole population. Furthermore, FACS analysis showed that serum-maintained cells sustained a higher level of receptor expression relative to serum-deprived cells. Additionally, HGF was found to stimulate proliferation, protein synthesis, and migratory responses. CONCLUSIONS: c-Met receptors are expressed in native epithelium, capsular bag cultures, and FHL124 cells. Receptor is distributed across the entire cell population; however, this expression is environmentally and mechanically sensitive. HGF is also present in capsular bags at all stages of culture. In addition, HGF can stimulate migration, proliferation, and protein synthesis. It therefore appears that a multifunctional autocrine loop involving HGF and c-met is in place and could be important in the development of posterior capsule opacification.     

miR-124-3p靶向调控KLF6基因表达对H2O2诱导的人晶状体上皮细胞增殖和凋亡的影响

目的　探讨微小RNA-124-3p（miR-124-3p）对H₂O₂诱导的人晶状体上皮细胞增殖及凋亡的影响及其靶向调控 Krüppel样因子6(Krüppel like factor 6,KLF6)的机制。方法　按HLE-B3细胞处理方式的不同,将其分为HLE-B3组、HLE-B3+ H₂O₂组、HLE-B3+H₂O₂+miR-NC组、HLE-B3+H₂O₂+miR-124-3p组、HLE-B3+H₂O₂+si-NC组、HLE-B3+H₂O₂+si-KLF6组、miR-124-3p+pcDNA3.1组、miR-124-3p+pcDNA3.1-KLF6组。利用MTT实验检测各组HLE-B3细胞增殖活性,流式细胞仪检测各组HLE-B3细胞凋亡。双荧光素酶报告基因实验验证miR-124-3p与KLF6的靶向关系。Western blot检测各组HLE-B3细胞中Cyclin D1、P21、Bax、Bcl-2蛋白表达。结果　H₂O₂可抑制人晶状体上皮HLE-B3细胞中miR-124-3p的表达（HLE-B3组0.79±0.07、HLE-B3+H₂O₂组0.31±0.03）（P＜0.05）,而明显促进KLF6 的表达;miR-124-3p过表达或抑制KLF6表达可促进HLE-B3细胞增殖,抑制HLE-B3细胞凋亡（19.34±1.27、7.66±0.38;19.29±1.33、11.46±1.02）,促进Cyclin D1（0.39±0.04、0.89±0.08;0.39±0.04、0.74±0.07）、Bcl-2表达（0.29±0.03、0.74±0.07;0.29±0.03、0.60±0.06）,抑制P21（0.73±0.07、0.26±0.03;0.79±0.07、0.33±0.03）、Bax表达（0.86±0.08、0.37±0.03;0.86±0.08、0.51±0.05）。共转染miR-124-3p mimics与WT-KLF6可明显降低HLE-B3细胞的荧光素酶活性（0.27±0.03、0.98±0.08）（P＜0.05）;过表达KLF6可逆转miR-124-3p对H₂O₂诱导的人晶状体上皮细胞HLE-B3细胞增殖及凋亡的作用。结论　miR-124-3p可通过靶向调控 KLF6表达进而促进H₂O₂诱导的人晶状体上皮细胞HLE-B3细胞增殖并抑制其凋亡。     

Effects of low and hyper Dk rigid gas permeable contact lenses on Bcl-2 expression and apoptosis in the rabbit corneal epithelium.

PURPOSE: To study Bcl-2 expression and apoptotic cell shedding of the rabbit corneal epithelium during extended wear of low and hyper Dk rigid gas permeable (RGP) contact lenses. METHODS: Rabbits were fit with either a low or a hyper Dk RGP lens (Dk/Ltotal= 10 and 97). The rabbits wore the lenses for either 24 hours, 3 days, or 1 week at which point they were humanely sacrificed. Immunocytochemistry and western blot analyses were performed to detect Bcl-2 in the corneal epithelium; TUNEL assay (TdT-mediated dUTP nick-end labeling) was used to identify apoptotic epithelial cells. RESULTS: 1) Immunocytochemistry: In the normal cornea, antibodies to Bcl-2 uniformly stained nuclei of all epithelial cell layers. Occasional surface epithelial cells, however, showed no anti-Bcl-2 nuclear staining; concomitant TUNEL assay revealed that all TUNEL-labeled-surface cells were Bcl-2 negative. By contrast, RGP contact lens wear, regardless of test lens oxygen transmissibility or lens wearing interval, significantly decreased both the total number of Bcl-2 negative and TUNEL-labeled cells on the epithelial surface (P < 0.05). In addition, contact lens wear was associated with labeling of keratocytes with TUNEL assay in the anterior stroma. 2) Western blot analysis: Total epithelial layer Bcl-2 expression was markedly decreased in the low Dk lens test group but was similar to control values in the hyper Dk lens test group. CONCLUSION: Bcl-2 protein seems to play an important role in the regulation of apoptotic cell shedding in the normal rabbit corneal epithelium. The identical staining pattern was seen in previous studies of the normal human cornea. RGP contact lens wear, however, appears to block the changes in Bcl-2 protein prior to apoptotic surface cell shedding, suggesting a lens-related anti-apoptotic effect. Taken together, these findings may explain why contact lens wear reduces surface cell exfoliation as previously reported in human studies.     

TGF beta-induced contraction is not promoted by fibronectin-fibronectin receptor interaction, or alpha SMA expression

PURPOSE: Transforming growth factor (TGF)-beta is a potent inducer of both transdifferentiation and contraction, which are regarded as critical processes that underpin tissue fibrosis. Consequently, transdifferentiation is believed to drive TGFbeta-mediated contraction. This study was conducted to determine the relationship between transdifferentiation of human lens epithelial cells and matrix contraction. METHODS: Real-time PCR was used to investigate gene expression of transdifferentiation markers in the human lens cell line FHL 124 and native lens epithelia. Contraction was assessed with a patch-contraction assay, whereby all areas covered by cells were measured with imaging techniques after fixation and cell staining with Coomassie blue. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. To prevent fibronectin-fibronectin receptor interaction 100 microM RGDS peptide was used. Suppression of TGFbeta-induced alphaSMA expression was mediated by siRNA technology. RESULTS: Real-time PCR analysis showed 10 ng/mL TGF-beta1 or -beta2 significantly increased expression of alphaSMA, fibronectin, and alpha5beta1 integrin (fibronectin receptor components) in FHL 124 cells and human lens epithelia. Cultures maintained in TGFbeta and RGDS showed a marked increase in the rate of contraction relative to TGF-beta alone. RGDS alone did not differ significantly from the control. Real-time PCR and Western blots showed reduced levels of message and alphaSMA protein when transfected with siRNA. alphaSMA knockdown did not prevent TGFbeta-induced contraction. CONCLUSIONS: A targeted inhibition approach demonstrated that key elements associated with transdifferentiation are not critical for TGFbeta-induced matrix contraction.