整合素-α5及其配体在大鼠角膜创伤愈合中的表达

整合素属细胞黏附分子，是由α和β亚单位通过非共价键形式结合成为跨膜糖蛋白，主要介导细胞与细胞和细胞与细胞外基质间的黏附，并在角膜创伤愈合中起重要作用。α5主要与β1结合。文献报道，配体纤维连接蛋白(fibronectin，FN)是以二聚体形式存在的大分子糖蛋白，经典受体为整合素-α5，大鼠角膜移植术后角膜创缘上皮细胞、迁移上皮细胞膜基底面及基质切口边缘α5及FN蛋白表达量明显增加；当创伤愈合后，其α5及FN蛋白表达消失。为此我们对大鼠角膜创伤愈合中整合素-α5及FN蛋白表达量进行检测，探讨其在角膜创伤愈合中的作用，现将结果报告如下。     

整合素在晶状体上皮细胞中的表达及其作用研究进展

整合素是介导细胞与细胞间及细胞与细胞外基质相互作用的重要黏附分子，其可参与晶状体上皮细胞的黏附和移行。本文综述了整合素在晶状体上皮细胞中的表达及其作用，以其对晶状体的发育、白内障及后发障的形成有进一步的了解。     

体外培养人晶状体上皮细胞整合素的表达

目的:研究体外培养人晶状体上皮细胞整合素的表达。方法:对人晶状体上皮细胞株SRA01/04进行培养并传代,经间接免疫荧光标记法处理细胞后上流式细胞仪检测各整合素的阳性表达率。结果:整合素α2,α3,α5,β1,β2在SRA01/04的阳性表达率分别为85.3%,97.6%,74.0%,97.7%,3.65%。整合素α3与β1之间无统计学差异(P>0.05),其余两两间比较均有显著统计学差异(P<0.01)。结论:人晶状体上皮细胞株SRA01/04整合素呈阳性表达。     

维拉帕米对人晶状体上皮细胞整合素表达的抑制作用

目的研究钙通道阻滞剂维拉帕米对人晶状体上皮细胞（LECs）株SRA01/04整合素表达的影响。方法对人LECs株SRA01/04进行培养并传代,分别以0.02、0.04、0.08mmol/L的维拉帕米作用于SRA01/0424h,经流式细胞仪检测SRA01/04各整合素的阳性表达率。结果整合素α2、α3、α5、β1、β2在人LECs株SRA01/04中呈阳性表达,维拉帕米对其表达具有抑制作用,并随药物浓度的增加而逐渐增强（P〈0.05）。结论钙通道阻滞剂维拉帕米对LECs整合素的表达具有抑制作用,可能阻碍整合素介导的LECs黏附、移行和增生,有望成为防治后发性白内障的有效药物。     

整合素与去整合素在后发性白内障方面的研究现状

晶状体摘除术后晶状体上皮细胞的增殖、黏附和迁移是后发性白内障的主要原因,整合素是一类介导C-C间以及C-ECM间的相互作用的黏附分子,在晶状体上皮细胞的增殖、黏附和迁移过程中发挥重要的作用,去整合素是一种小分子多肽,它可以特异性的被整合素识别并黏附,从而抑制整合素的生物学作用,本文就整合素的功能,整合素在晶状体上皮细胞上的分布及功能,晶状体上皮细胞与后发性白内障之间的关系以及去整合素在后发性白内障防治方面的研究进展做一综述。     

去整合素对人眼晶状体上皮细胞的粘附移行作用

目的研究去整合素对体外培养的人眼晶状体上皮细胞生长的抑制作用。方法对先天性白内障患者术中环行撕前囊膜,进行原代培养并传代,取第2代细胞用于实验。抗粘附实验:将悬浮的细胞与不同浓度的去整合素(5μg·L-1、10μg·L-1、20μg·L-1、40μg·L-1、80μg·L-1)在37℃、50mL·L-1CO2条件下一起孵育30min。然后接种于涂有鼠尾胶原的96孔培养板。8h后,用HBSS轻洗各孔2次,MTT法测定粘附细胞的吸光度。抗移行实验:对融合的细胞进行划线,80μg·L-1的去整合素进行干预,24h、48h、72h后,细胞计数法观察裸露区内细胞的移行数量。结果不同浓度的去整合素(20μg·L-1、40μg·L-1、80μg·L-1)对晶状体上皮细胞的粘附均具有抑制作用且呈剂量依赖性。与对照组相比,24h、48h、72h内80μg·L-1的去整合素可显著抑制晶状体上皮细胞的移行。结论去整合素抑制体外培养的人眼晶状体上皮细胞的粘附及移行。     

AG490对高糖环境中人晶状体上皮细胞向间充质转化的抑制作用

目的:研究在高糖诱导人晶状体上皮细胞向间充质转化的过程中JAK-STAT通路的作用及AG490对此过程的影响。方法:低糖(5.5mmol/L)DMEM培养液体外培养人晶状体上皮细胞系SRA01/04。高糖(30.5mmol/L)处理细胞,按照是否加入AG490及其浓度的不同分为实验组和对照组。CCK-8试剂盒检测晶状体上皮细胞的活性,选择实验组AG490浓度为10μmol/L和50μmol/L,处理时间分别为6,12,24和48h;细胞划痕实验检测细胞迁移能力的变化;RT-PCR方法半定量检测TGF-β1,FN和α-SMA的mRNA表达量变化。结果:在不同的处理时间(6,12,24,48h),高糖组(HG组)与低糖组相比,细胞的迁移能力增加(P<0.05),TGF-β1,FN和α-SMA表达量增高(P<0.05);AG490组与HG组相比,细胞的迁移能力降低(P<0.05),TGF-β1,FN和α-SMA的mRNA表达量下降。结论:JAK-STAT通路通过影响TGF-β1和细胞外基质转录表达参与了高糖诱导的人晶状体上皮细胞向间充质转化的过程。JAK抑制剂AG490对此过程有抑制作用,且随着AG490浓度的增加其抑制作用增强。     

整合素在角膜上皮创伤愈合中的研究进展

整合素作为一类重要的细胞黏附分子,通过影响细胞的形态,介导细胞的黏附、迁移和增生,在角膜上皮创伤愈合中发挥了重要的作用。讨论α2β1、α3β1、α5β1、αvβ3、α6β4、α9β1和αvβ6这7种整合素在角膜上皮创伤愈合中的研究进展及其临床意义。α6β4整合素为半桥粒的主要组成部分,介导角膜上皮细胞在细胞外基质上的静态黏附,损伤后该黏附就转变为α2β1、α3β1整合素介导的动态黏附,细胞在黏附-去黏附的过程中实现迁移,从而修复创面。α6β4、α3β1整合素相互协调作用,实现上皮细胞的板层状运动。研究还发现α6β4、α3β1整合素的活化还能促进细胞的增生。损伤后上皮细胞表面α5β1、αvβ3整合素的表达上调,二者与黏着斑的形成密切相关。α9β1和αvβ6为近年来新发现的与角膜上皮创伤愈合有关的整合素,其具体作用尚有待进一步的研究。     

整合素在真菌与角膜上皮细胞黏附过程中的表达

目的 探讨不同真菌菌种与人角膜上皮细胞黏附过程中整合素的表达及其作用机制.方法 实验研究.体外培养人角膜上皮细胞系,建立茄病镰刀菌(CGMCC 3.1829)和烟曲霉菌(CGMCC 3.0772)与角膜上皮细胞黏附的体外模型.茄病镰刀菌或烟曲霉菌与人角膜上皮细胞共孵育后,用无菌的磷酸盐缓冲溶液冲洗掉未黏附的真菌.提取细胞的总RNA,反转录为cDNA后采用实时荧光定量聚合酶链反应(RT-PCR)检测不同时间点上不同真菌与角膜上皮细胞黏附的14种整合素分子mRNA水平的表达.各时间点之间基因表达差异的比较采用单因素方差分析.结果 在烟曲霉菌与人角膜上皮细胞黏附的过程中,随黏附时间延长,整合素家族白细胞黏附受体组成员编码基因整合素αL(ITGAL)、整合素α型(ITGAM)、整合素αX(ITGAX)及整合素β2(ITGB2)的表达显著上调.其中ITGAL的表达最高上调2倍(F=29.39,P<0.01),ITGAM的表达最高上调4倍(F=20.26,P<0.01),ITGAX的表达最高上调2.5倍(F=2.51,P<0.05),ITGB2的表达最高上调3.4倍(F=3.923,P<0.05).而在茄病镰刀菌与人角膜上皮细胞黏附的过程中,此14种整合素的表达未见显著差异.结论 整合素家族白细胞黏附受体组(β2组)成员αLβ2、αMβ2及αXβ2均参与烟曲霉菌与角膜上皮细胞的黏附;未见茄病镰刀菌与角膜上皮细胞的黏附过程中整合素表达的差异.整合素介导的黏附因菌种而异.     

去整合素对人眼品状体上皮细胞的粘附移行作用

目的研究去整合素对体外培养的人眼晶状体上皮细胞生长的抑制作用。方法对先天性白内障患者术中环行撕前囊膜,进行原代培养并传代,取第2代细胞用于实验。抗粘附实验:将悬浮的细胞与不同浓度的去整合素(5μg·L-1、10μg·L-1、20μg·L-1、40μg·L-1、80μg·L-1)在37℃、50mL·L-1CO2条件下一起孵育30min。然后接种于涂有鼠尾胶原的96孔培养板。8h后,用HBSS轻洗各孔2次,MTT法测定粘附细胞的吸光度。抗移行实验:对融合的细胞进行划线,80μg·L-1的去整合素进行干预,24h、48h、72h后,细胞计数法观察裸露区内细胞的移行数量。结果不同浓度的去整合素(20μg·L-1、40μg·L-1、80μg·L-1)对晶状体上皮细胞的粘附均具有抑制作用且呈剂量依赖性。与对照组相比,24h、48h、72h内80μg·L-1的去整合素可显著抑制晶状体上皮细胞的移行。结论去整合素抑制体外培养的人眼晶状体上皮细胞的粘附及移行。     

tTG小干扰RNA对人晶状体上皮细胞转分化和细胞外基质沉积的抑制作用

目的探讨RNA干扰抑制组织型转谷氨酰胺酶(tissue transglutaminase,tTG)的表达对人转化生长因子β2(transforming growth factor-beta2,TGF-β2)诱导的晶状体上皮细胞(lens epithelial cells,LEC)转分化和细胞外基质沉积的抑制作用。方法设计并合成针对tTG的3对小干扰RNA(siRNA):tTG-siRNA-1、tTG-siRNA-2、tTG-siRNA-3,用Real-time PCR和Western blot检测转染了siR-NA后HLE-B3细胞表达tTG mRNA和tTG蛋白的变化。然后将体外培养的HLE-B3细胞分为正常对照组、TGF-β2组和TGF-β2+siRNA组。48h后,用Western blot检测各组tTG、平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(fibronec-tin,FN)和Ⅳ型胶原(collagenⅣ,Col-Ⅳ)的表达。结果转染tTG-siRNA-1、tTG-siRNA-2、tTG-siRNA-348h后,tTG mRNA的表达分别下降为空白对照组的46.60%、12.84%、66.75%(均为P<0.05);tTG蛋白的表达量分别下降为空白对照组的60.49%、27.87%、55.91%(均为P<0.01)。Real-time PCR与Western blot检测结果一致显示了tTG-siRNA-2对tTG基因的抑制效果最佳。与正常对照组相比,TGF-β2组中tTG、α-SMA、FN、Col-Ⅳ的表达量均明显增加(均为P<0.01);与TGF-β2组相比,TGF-β2+tTG-siRNA-2组中tTG、α-SMA、FN、Col-Ⅳ的表达量均明显减少(均为P<0.01)。结论靶向tTG的siRNA可以显著降低tTG的表达水平,同时可以明显抑制TGF-β2诱导的HLE-B3细胞合成α-SMA、FN、Col-Ⅳ。提示tTG是介导TGF-β2诱导人LEC转分化和细胞外基质沉积的重要分子。     

Mechanisms of inhibition of elemene on human lens epithelial cell proliferation in vitro

AIM: To study the effects of elemene (Ele) on proliferation and cell cycle of human lens epithelial cells B3 (HLE-B3) and the mechanisms of its signal transduction. METHODS: Recombinant human basic fibroblast growth factor (rhbFGF) was used to induce proliferation of HLE-B3 cells, which were incubated with 80mg/L Ele for 24 hours. The inhibitory effects of Ele on the proliferation of HLE-B3 cells were evaluated by MTT method. The effect of Ele on HLE-B3 cell cycle was analyzed by flow cytometry(FCM). The expressions of protein kinase A (PKA) and protein kinase G (PKG) of HLE-B3 were also analyzed by FCM. RESULTS: Ele altered the cell cycle of HLE-B3 and effectively inhibited HLE-B3 cell proliferation induced by rhbFGF. Ele up-regulated PKA and down-regulated the expression of PKG in HLE-B3 cell. CONCLUSION: Ele inhibits HLE-B3 proliferation, making it an attractive potential agent in regimens to treat after- cataracts.     

中药单体榄香烯体外对人晶状体上皮细胞内胶原蛋白合成的影响

目的:探讨中药单体榄香烯(effects of elemene,Ele)对人晶状体上皮细胞系(human lens epithelial cells B3,HLE-B3)内Ⅰ型胶原蛋白和Ⅲ型胶原蛋白合成的影响。方法:利用10μg/L碱性成纤维细胞生长因子(recombinant human basic fibroblast growth factor,rhbFGF)诱导HLE-B3增殖,将80mg/L的Ele作用在处于增殖状态下的HLE-B3,24h后采用双抗体夹心ABC-酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测Ele作用后HLE-B3内Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达。结果:rhbFGF作用后HLE-B3内Ⅰ型胶原蛋白浓度为53.5±5.4μg/L,较正常组(38.5±2.3μg/L)明显升高,Ele作用后HLE-B3内Ⅰ型胶原蛋白的浓度为29.5±2.9μg/L,较rhbFGF组明显下降(P<0.01)。rhbFGF作用后HLE-B3内Ⅲ型胶原蛋白浓度为1.27±0.29μg/L,较正常组(0.83±0.12μg/L)明显升高,Ele作用后HLE-B3内Ⅲ型胶原蛋白的浓度为0.69±0.11μg/L,较rhbFGF组明显下降(P<0.01)。结论:Ele抑制rhbFGF诱导的HLE-B3增殖的同时也能抑制HLE-B3内Ⅰ,Ⅲ型胶原蛋白合成,干扰HLE-B3纤维化,可望成为防治后囊膜混浊的理想药物。     

反义c-myc基因转染抑制人晶状体上皮细胞增殖的研究

目的探讨腺病毒介导的反义cmyc基因(AdASmyc)转染对人晶状体上皮细胞(HLEC)系B3(HLEB3)增殖、凋亡及细胞周期的影响。方法将AdASmyc转染HLEB3,观察细胞形态变化;采用生长曲线法、噻唑蓝比色法分析细胞增殖改变;使用流式细胞仪测定细胞凋亡变化、分析细胞周期改变(DNA倍体法);采用逆转录聚合酶链反应(RTPCR)法、Western免疫印迹法检测细胞cmycmRNA及其蛋白产物表达的变化。结果AdASmyc成功转染后,HLEB3逐渐变圆、脱壁。转染后24~96h细胞的增殖受到抑制,与相应对照组比较,差异均有统计学意义(P<001);凋亡细胞比例(转染48h为1833%,转染96h为2693%)明显升高,与相应对照组(48h为319%,96h为175%)比较,差异均有统计学意义(P<001);细胞出现G1期阻滞现象,表现为G1期细胞比例[转染48h为(6050±759)%,转染96h为(8190±860)%]增加和S期细胞比例[转染48h为(2840±338)%,转染96h为(1675±897)%]下降,与相应对照组比较,差异均有统计学意义(P<001);RTPCR法和Western免疫印迹法检测发现cmycmRNA及其蛋白产物水平特异性下调。结论AdASmyc可成功转染HLEB3,并特异性抑制cmyc基因的表达,诱发细胞G1期阻滞,有效抑制细胞增殖,诱导细胞凋亡。     

组织型转谷氨酰胺酶抑制剂对TGF—β2诱导人LECs表达纤维连接蛋白和Ⅳ型胶原的影响

目的观察组织型转谷氨酰胺酶（tTG）特异性抑制剂（MDC）对TGF—β2诱导人晶状体上皮细胞（LECs）表达纤维连接蛋白（FN）、Ⅳ型胶原（Col-Ⅳ）的影响。方法将含10％胎牛血清的DMEM培养基中培养的LECs株HLE—B3传代后分为5组：无血清培养基培养的HLE—B3为正常对照组;加入10μg／LTGF—β2处理的HLE—B3为TGF—β2组;10μg／LTGF—β2与100、200、400μmol／LMDC共同处理的HLE—B3为不同浓度MDC组。用半定量RT—PCR检测tTG、FN、Col-Ⅳ在HLE—B3中的表达,计算A（tTG／β-actin）、A（FN／β-actin）、A（Col-Ⅳ／β-actin）值。结果正常体外培养的HLE-B3中可表达一定量的tTG、FN及Col-Ⅳ。与正常对照组相比,10Ixg／LTGF-β2组中tTG、FN、Col—Ⅳ的表达明显增加（t=33．95,P〈0．01;t=38．24,P〈0．01;t=13．48,P〈0．01）;与TGF—B,组相比,100、200、400μmol／LMDC组中FN和Col-Ⅳ的表达明显减少（P〈0．01）。100、200、400μmol／LMDC组各组间FN和Col—Ⅳ的表达差异均有统计学意义（P〈0．01）。结论MDC可剂量依赖性地抑制TGF—β2诱导的LECs中FN、Col—Ⅳ表达的增加。tTG可促进人LECs表达FN、Col—Ⅳ,并参与后囊膜混浊（PCO）的形成。     

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。     

An in vitro study of human lens epithelial cell adhesion to intraocular lenses with and without a fibronectin coating

PURPOSE: To demonstrate differences in human lens epithelial cell adhesion to different intraocular lens biomaterials in vitro and to determine whether these differences can be influenced by coating the intraocular lens surface with commercially available fibronectin. METHODS: A prospective laboratory-based study comparing human lens epithelial cell adhesion to silicone (n=18), polymethylmethacrylate (PMMA; n=18), and acrylic (n=18) intraocular lenses in vitro. The three types of intraocular lenses were then coated with fibronectin: silicone (n=6), PMMA (n=6), and acrylic (n=6). The main outcome measure was the mean number of lens epithelial cells attached to each lens type after 24 hours of incubation. RESULTS: In the uncoated lens group, there was a significantly higher number of lens epithelial cells attached to the acrylic than to the silicone or PMMA lenses (P<0.001). Coating the lenses with fibronectin caused a significant increase in attachment of lens epithelial cells for all three lens types. CONCLUSIONS: There was a significant difference in the degree of lens epithelial cell attachment to the various types of intraocular lenses in vitro. Cell attachment was more prominent in the acrylic lenses, but the fibronectin coating negated differences in lens type and caused a significant increase in cell attachment across all groups.     

The effects of extracellular matrix on cell attachment, proliferation and migration in a human lens epithelial cell line

Lens capsule consists of several kinds of extracellular matrix (ECM) which may play an important role in cell attachment, migration and proliferation of lens epithelial cells as a basement membrane. We have investigated the effects of ECM on cell attachment, proliferation and migration in a human lens epithelial (HLE) cell line. The HLE cell line, SRA 01/04, which was transfected with large T-antigen of SV40 was cultured in the absence of serum. Culture plates were coated with human type IV collagen, laminin or fibronectin. The number of cells were counted at 30-180 min and 3, 5 and 7 days of culture. The rate of BrdU incorporation was measured to study the cell proliferation. The cell migration was measured 1, 3, 5 and 7 days after seeding cells. Integrins, the receptors of ECM, were also detected using antibodies for the cell membrane antigens (CD49b, CD49c, CD49e) by an immunohistochemical method. Although less than 10% of cells attached to the non-coated plate and 50-60% of cells attached to the ECM-coated plates, there was no difference of cell attachment among each ECM used. The cell attachment was almost complete during the first 30 min of culture. Cell proliferation was not enhanced, but cell survival was aided by culture on the ECM components for up to 7 days. The area of cell attachment enlarged on the ECM-coated plates, whereas no migration was observed on the non-coated plate. These data indicate that ECM is the essential factor for cell attachment and increases migration of HLE cells.     

Comparative effects of TGF-β2/Smad2 and TGF-β2/Smad3 signaling pathways on proliferation, migration, and extracellular matrix production in a human lens cell line

The signaling pathway of transforming growth factor β2 (TGF-β2)/Smad plays an important role in the pathological process in posterior capsule opacification (PCO) after cataract surgery. Smad2 and Smad3 are both receptor-regulated Smads (R-Smads) of the TGF-β2 signaling pathway. We aim to find which among Smad2, Smad3, and Smad2&3 plays a key role in PCO pathology. The signal characteristics of TGF-β2 and Smad proteins in the human lens cell line HLE-B3 were investigated. Smad2, Smad3, or Smad2&3 were silenced using small interfering RNA. We then tested cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell growth curve assays, migration by transwell and wound-healing assays, and extracellular matrix production including α-smooth muscle actin (αSMA), fibronectin, and type I collagen by real-time PCR assay, with and without TGF-β2 exposure. Silencing Smad3 blocked the effect of TGF-β2 on cell proliferation and production of fibronectin and type I collagen. Silencing Smad2 blocked the effect of TGF-β2 on cell migration and production of αSMA. Smad2 depletion enhanced Smad3 activity in cell proliferation and ECM production, whereas Smad3 depletion enhanced Smad2 activity in migration and αSMA expression. Silencing Smad2 and Smad3 efficiently blocked the effect of TGF-β2on cell proliferation, migration, and extracellular matrix production. Smad2 and Smad3 are both key in the TGF-β2 signaling pathway. We can prevent the development of PCO following cataract surgery by blocking the TGF-β2/Smad2&3 signaling pathway.     

Induction of apoptosis by the calcium antagonist mibefradil correlates with depolarization of the membrane potential and decreased integrin expression in human lens epithelial cells

Background Posterior capsule opacification is still the major complication in cataract surgery and is caused by migration and proliferation of residual lens epithelial cells. The challenge of a suitable therapy to inhibit capsule opacification is to specifically interfere with cellular mechanisms. Our approach using the T-calcium channel antagonist mibefradil is based on the hypothesis that this drug inhibits the signaling pathways mediated by cell adhesion.Methods The influence of mibefradil dihydrochloride was investigated on primary human lens epithelial cells (hLEC) from cataract surgery and on the human lens cell line HLE-B3. Apoptosis was quantitatively analyzed by flow cytometry (% increase of the sub-G1 peak), and verified by confocal microscopy (annexin V-biotin, TUNEL reaction). The membrane potential was detected by a membrane potential-sensitive dye. Integrin expression and proliferation were measured by flow cytometry. T-calcium channels in hLEC were verified by the whole-cell configuration of the patch-clamp technique.Results Mibefradil induced apoptosis in hLEC. Early signs of apoptosis were observed after only 4 h of incubation with mibefradil, accompanied by a significantly reduced cell area. Apoptosis correlated with inhibited integrin expression, reduced proliferation and the depolarization of the membrane potential. We could identify calcium channels of the T-type in our primary hLEC.Conclusions We suggest that depolarization of the membrane potential and the inhibition of integrin expression leads to the loss of cell adhesion, which is the reason for the induction of apoptosis. Thus, mibefradil seems to be a suitable drug to prevent cell adhesion, migration and proliferation.