糖尿病兔晶状体后囊膜混浊模型的建立

背景实验性糖尿病动物模型的制作是对糖尿病性眼病进行实验研究的关键环节,目前国内普遍应用的方法是链脲佐菌素和四氧嘧啶（ALX）注射,但前者价格昂贵,后者造模过程中动物的死亡率较高。目的探讨ALX注射制作糖尿病兔晶状体后囊膜混浊（PCO）模型并降低死亡率的方法,并观察高血糖对晶状体PCO形成的早期影响。方法将清洁级健康新西兰雄性大白兔40只随机分为2组;其中20只兔经耳缘静脉一次性注射ALX90mg／kg建立糖尿病模型作为高血糖组,另20只兔以同样的方法注射等量生理盐水作为正常血糖组。药物注射后2周时高血糖组兔血糖升高到12．0mmol／L以上可判断为建模成功,2组分别行兔右眼透明晶状体囊外摘出术并对晶状体PCO进行分级。于术后第6、10、14天取眼球,应用免疫组织化学法观察增生细胞核抗原（PCNA）在后囊膜晶状体上皮细胞（LECs）中的表达情况。结果ALX注射后糖尿病兔的成模率为70％。术后第6、10、14天时,高血糖组兔体质量均明显低于正常血糖组,但血糖明显高于正常血糖组,差异均有统计学意义（P〈0．05）。高血糖组术后14d时观察的3只兔中,2只兔出现后囊膜2级混浊,另1只兔出现1级混浊。正常血糖组术后14d时观察的3只兔后囊膜均出现1级混浊。免疫组织化学染色显示,术后第10天高血糖组可见PCNA在LECs的细胞核中表达,但正常血糖组未见PCNA的表达。术后第14天时高血糖组PCNA增生指数为0．86±0．04,明显高于正常血糖组的0．25±0．03,差异有统计学意义（t=-16．171,P=0．000）。结论90mg／kg的ALX静脉注射能形成稳定的糖尿病兔PCO模型;高血糖是促进PCO发生发展的重要因素之一。     

直角边缘人工晶状体预防兔后囊膜混浊的实验研究

目的探讨直角边缘人工晶状体（intraocular lens，IOL）预防后囊膜混浊（posterior capsule opacification，PCO）的作用。方法30只新西兰兔进行超声乳化晶状体摘出联合囊袋内IOL植入术后，随机植入Crane OV-55CP、Crane OV-55C、Alcon TYPE 5C 3种IOL之一。观察术后并发症和PCO情况。术后3月行光镜和透射电镜检查，观察晶状体后囊膜的形态学变化。结果术后3月Crane OV-55CP组的PCO程度比Crane OV-55C和Alcon TYPE 5C组轻（P〈0．05），各组Soemmefing环形成程度无差异（P〉0．05）。病理学检查发现Crane OV-55CP组兔赤道部增生的晶状体上皮细胞在人工晶状体的直角边缘处受到了阻挡。Crane OV-55C和Alcon TYPE 5C组大量晶状体上皮细胞迁移至后囊膜。结论直角边缘IOL延缓了兔PCO的发生、发展，是预防PCO简便安全有效的方法。     

无防腐剂的1%利多卡因对兔晶状体上皮细胞影响的组织病理学研究

目的探讨无防腐剂的1％利多卡因对兔晶状体上皮细胞（LECs）的影响,为寻求白内障术中清除上皮,预防囊膜混浊的有效药物提供实验依据。方法采集29只兔（58只眼）晶状体前囊膜及赤道部囊膜标本,分为3组,对照组囊膜不加药物处理、BSS组囊膜用BSS浸泡1min、利多卡因组囊膜用利多卡因浸泡1min,后行锥虫蓝-茜素红染色,显微镜照相,观察LECs活性同时对死亡细胞进行计数;并通过HE染色及透射电镜观察兔LECs的组织病理学改变。锥虫蓝．茜素红染色及死亡细胞计数15只兔（30只眼）：其中对照组5只兔（10只眼）,BSS组5只兔（10只眼）,利多卡因组5只兔（10只眼）;HE染色9只兔（18只眼）：其中对照组1只兔（2只眼）,BSS组4只兔（8只眼）,利多卡因组4只兔（8只眼）;透射电镜5只兔（10只眼）：其中对照组1只兔（2只眼）,BSS组2只兔（4只眼）,利多卡因组2只兔（4只眼）。结果对照组兔晶状体前囊膜上皮细胞死亡率（1．51±0．39）％,赤道部囊膜上皮细胞死亡率（1．52±0．32）％;BSS组兔晶状体前囊膜上皮细胞死亡率（1．78±0．50）％,赤道部囊膜上皮细胞死亡率（1．77±0．47）％;利多卡因组兔晶状体前囊膜上皮细胞死亡率（13．01±4．67）％,赤道部囊膜上皮细胞死亡率（9．59±3．35）％。经嵌套试验的方差分析,利多卡因组细胞死亡率明显高于对照组与BSS组（P〈0．05）。HE染色显示对照组与BSS组兔LECs仍贴附于囊膜上,未见明显病理改变。利多卡因组部分细胞与囊膜间产生空隙,并从囊膜上脱落,细胞空泡变性。透射电镜观察下对照组与BSS组细胞间及细胞与囊膜间连接完整。利多卡因组部分细胞间及细胞与囊膜间的连接被破坏,细胞脱落较多,排列松散。细胞膜塌陷,胞质内有大量的空泡形成,细胞结构被破坏。结论无防腐剂的1％利多卡因能松解兔LECs间及细胞与晶状体囊膜间的连接,并可导致LECs变性、死亡。     

去整合素kistrin对兔眼晶状体后囊Ⅳ型胶原表达的影响

背景 白内障囊外摘出术后残留的晶状体上皮细胞(LECs)增生是后囊膜胶原产生进而形成后发性白内障的生物学基础,去整合素可与细胞外基质(ECM)竞争结合整合素分子,理论上可以防治后发性白内障的形成,但其具体的作用机制有待进一步研究. 目的 研究去整合素kistrin对兔眼晶状体后囊Ⅳ型胶原表达的影响.方法24只新西兰大白兔按照随机数字表法分为kistrin注射组及生理盐水对照组,每组12只.两组兔均行右眼透明晶状体囊外摘出术,建立兔晶状体后囊膜混浊(PCO)模型,术毕kistrin注射组囊袋内注入80 mg/L kistrin 0.2 ml,生理盐水对照组注入等量生理盐水.术后1、3、5、7、14 d,在裂隙灯下观察实验动物晶状体PCO情况,并按照Odrich法进行分级.术后14d及3个月分别处死两组兔各6只,取出晶状体进行常规石蜡切片,行苏木精-伊红染色,光学显微镜下观察晶状体病理改变;行Masson染色观察晶状体囊袋内胶原纤维增生情况,免疫组织化学法检测兔晶状体后囊Ⅳ型胶原的表达. 结果 术后14 d,生理盐水对照组与kistrin注射组各级PCO的眼数差异无统计学意义(P=0.093),术后1、2、3个月,生理盐水对照组形成2～3级PCO的眼数明显多于kistrin注射组,差异均有统计学意义(P=0.041、0.014、0.022).晶状体组织学检查表明,术后14 d生理盐水对照组LECs层数明显多于kistrin注射组,瞳孔区后囊膜可见单层细胞黏附,kistrin注射组后囊则保持光滑,术后3个月可见生理盐水对照组晶状体后囊的LECs转化为纤维细胞,kistrin注射组较少见.Masson染色显示术后3个月生理盐水对照组晶状体前囊膜撕囊口处与后囊膜之间胶原纤维的蓝绿色染色明显多于kistrin注射组.免疫组织化学染色表明,术后14 d及30 d,生理盐水对照组晶状体后囊膜Ⅳ型胶原的灰度值均明显低于kistrin注射组,差异均有统计学意义(P=0.000、0.001). 结论 去整合素kistrin能够抑制兔眼晶状体囊外摘出术术后晶状体后囊LECs和Ⅳ型胶原增生.     

胰蛋白酶复合黏弹剂对后发性白内障的预防作用

目的探讨胰蛋白酶复合黏弹剂预防后发性白内障（PCO）的有效性及安全性。方法将25只新西兰白兔50只眼平均分为5组,每组10只眼。所有实验眼均行白内障超声乳化手术,在晶状体囊袋内分别注入普通黏弹剂、质量分数0.5%胰蛋白酶复合黏弹剂＋血清复合黏弹剂、0.75%胰蛋白酶复合黏弹剂＋血清复合黏弹剂、1%胰蛋白酶复合黏弹剂＋血清复合黏弹剂或1%胰蛋白酶复合黏弹剂并在囊袋内保留3min。摘除实验眼并制备常规固定切片,行苏木精-伊红染色,光镜下观察各组兔晶状体上皮细胞（LECs）和角膜内皮细胞（CECs）的形态学变化。结果 LECs计数随着胰蛋白酶剂量的增加而逐渐减少,4个实验组LECs计数的差异有统计学意义（H=36.696,P=0.00）。1%胰蛋白酶复合黏弹剂＋血清复合黏弹剂组的CECs计数为（34.0±2.6）个,而1%胰蛋白酶复合黏弹剂组为（27.0±4.7）个,2组差异有统计学意义（Z=-3.792,P=0.00）。组织学检查表明,光学显微镜下普通黏弹剂组LECs结构和排列正常;0.5%胰蛋白酶复合黏弹剂＋血清复合黏弹剂组和0.75%胰蛋白酶复合黏弹剂＋血清复合黏弹剂组可见部分LECs脱落,眼内其他组织形态无明显异常;1%胰蛋白酶复合黏弹剂＋血清复合黏弹剂组LECs完全脱落,但CECs及眼内其他组织形态无明显异常;1%胰蛋白酶复合黏弹剂组LECs完全脱落,伴有CECs的小片状脱落。结论白内障术中运用1%胰蛋白酶复合黏弹剂配合血清复合黏弹剂能安全有效地去除LECs,为PCO的预防提供了一个新的治疗途径。     

多聚赖氨酸-乙二胺四乙酸交联物抑制兔晶状体上皮细胞生长的研究

背景后囊膜混浊（PCO）是现代白内障囊外摘出术后引起视力下降的主要原因。研究证实多聚赖氨酸-乙二胺四乙酸（EDTA）交联物（PLE）能够降低兔PCO的发生率。目的研究PLE对体外培养的兔晶状体上皮细胞（LECs）生长的抑制作用及有效药物浓度。方法取3月龄新西兰白兔晶状体前囊膜进行体外植块培养获得兔LECs并进行传代,取第2～3代传代培养的LECs消化后按1×10^5个／ml密度接种于96孔培养板中。在培养皿中加入12．5、25．0、50．0、100．0μmol／L的PLE,培养皿中加入DMSO培养液作为对照组。PLE作用48h用MTT比色法测定PLE对兔LECs增生的抑制作用并计算各浓度PLE组的吸光度（A490）值及其抑制率。结果≤25．0μmol／LPLE组可见细胞生长及形态改变不明显。≥50．0μmol／LPLE各组可见细胞生长及形态有明显改变,增生缓慢,生长差,数量减少,贴壁能力减弱。12．5、25．0、50．0、100．0μmol／L的PLE组LECs的A490值分别为0．278±0．013、0．266±0．028、0．260±O．022和0．247±0．012,均明显低于DMSO对照组的O．311±0．038（P=0．035、0．011、0．009、0．013）,且呈明显的剂量一效应依赖关系。12．5、25．0、50．0、100．0μmol／LPLE组对兔LECs的抑制率分别为10．61％、14．47％、16．40％和20．58％。结论PLE可以浓度依赖的方式抑制兔LECs的生长,可为临床筛选出防治PCO的药物提供依据。     

儿童先天性白内障不同术式后发性白内障形成的病理学分析

目的 探讨预防儿童后发性白内障的手术方式.方法 回顾性研究先天性白内障患儿50例69眼,按不同手术方式(超声乳化晶状体吸出术、超声乳化晶状体吸出联合后囊膜连续环形撕囊术及超声乳化晶状体吸出联合后囊膜连续环形撕囊及前部玻璃体切割术)分为Ⅰ、Ⅱ、Ⅲ 3组,术后随访3～48个月,对各组术眼进行眼底镜、裂隙灯、组织病理学及电镜检查.结果 3组术眼晶状体后囊膜周边部均可见残留晶状体上皮细胞(lens epithelial cells,LECs)向梭形成纤维细胞转化,并可见残留的LECs形成半透明的球形Elschnig 珍珠小体;晶状体前囊膜亦可见少量残留的LECs及Elschnig珍珠小体.Ⅰ组后囊膜中央部可见多量增生的成纤维细胞及LECs;Ⅱ组后囊膜撕囊孔缘处可见成纤维细胞及排列紊乱的LECs,术眼视区中央部的增生膜内可见多量的羽毛状胶原原纤维;Ⅲ组视区中央无增生膜形成,后囊膜撕囊孔缘仅见少量残留LECs.3组中央视区后发性白内障发生率分别是:Ⅰ组 43.48%,Ⅱ组17.39%,Ⅲ组0,Ⅲ组多数术眼中央视区清亮,后囊孔缘处未见明显病理学改变.结论 儿童先天性白内障摘出术中,行后囊连续环形撕囊 前部玻璃体切割术可有效预防儿童中央视区后发性白内障的发生.     

EDTA与多聚赖氨酸的铰链物抑制兔后发性白内障的实验研究

目的：探讨EDTA与多聚赖氨酸的铰链物对兔后发性白内障的防治作用。方法：将20只新西兰白兔40眼随机分为A,B,C,D共4组,4组均行透明晶状体囊外摘除术。A组为对照组,术中灌注液为BSS;B,C,D组为治疗组,术中灌注液分别为浓度为10mg/L的EDTA、多聚赖氨酸、EDTA与多聚赖氨酸的铰链物的BSS溶液。2mo后行晶状体后囊膜切片,HE染色统计晶状体上皮细胞（LECs）的密度;并行免疫组织化学染色,用医学图象分析系统检测PCNA表达平均光密度（灰度值OD）。结果：经HE染色,后囊膜LECs密度B和D组较A和C组少,且D组的LECs密度小于B组,均有显著性差异（P〈0.01）;A和C组的LECs密度相差不大,无统计学差异（P〉0.05）。免疫组织化学进行平均光密度测定,A和C组PCNA表达强阳性,B组呈部分阳性表达,D组阳性表达较B组更少。B和D组与A组、B组与D组均有显著性意义（P〈0.01）。A组和C组无显著差异性（P〉0.05）。结论：在活体兔眼中,EDTA、多聚赖氨酸与EDTA的铰链物均有抑制兔LECs增殖的作用,且EDTA与多聚赖氨酸的铰链物组对LECs的抑制作用优于EDTA组,多聚赖氨酸组对LECs的抑制作用不明显。     

BALB/c小鼠后发性白内障动物模型的建立和观察

目的建立BALB／c小鼠后发性白内障（posterior capsule opacification，PCO）动物模型并检测Sox1／2胚胎晶状体发育调控基因在PCO中的表达。方法腹腔麻醉联合表面麻醉下对30只BALB／c小鼠行右眼晶状体囊外摘出术，分别于术后即刻、3d、1周、2周和1个月对术眼进行裂隙灯显微镜及组织病理学检查，观察PCO形成的时间、部位、发展过程及组织形态学改变；采用逆转录聚合酶链反应（RT-PCR）方法检测Sox1／2胚胎晶状体发育调控基因在术后不同时间点PCO中的表达。结果裂隙灯显微镜观察：后囊膜皱褶、混浊由周边部向中央区发展伴Elschnig小体和晶状体纤维生成，其程度随时间推移日渐加重；再生晶状体形态和大小与正常晶状体相似但透明度明显下降。组织病理学检查：手术后即刻，赤道部和前囊膜下可见单层晶状体上皮细胞（lens elial cell，LEC），后囊膜表面无LEc及晶状体皮质残留；术后3d，赤道部LEC增生并迁移至后囊膜。囊袋周边部LEC开始早期纤维分化，但核仍靠近后囊膜表面；术后1周，赤道部LEC继续分化，细胞伸长呈带状伴核远离后囊膜表面；术后2周，周边部晶状体纤维细胞持续增多，形成与正常晶状体赤道部形态类似的弓形带；术后1个月。新生晶状体纤维几乎填充整个残余囊袋，排列欠规则，细胞核罕见。RT-PcR检测：术后3d、1周、2周及1个月的PC0组织中可检测到Sox1／2条带；术后即刻囊袋组织中无Sox1／2表达。结论BALB／c小鼠可成功建立PCO动物模型并检测到Sox1／2胚胎晶状体发育调控基因的表达，为在分子生物学水平上进一步探索PCO的发病机制提供了有利条件，具有重要的应用价值。【眼科新进展2007；27（2）：91-95]     

去整合素Echistatin对糖尿病兔早期后发性白内障的抑制作用及眼内安全性

背景 后囊膜混浊（PCO）是影响白内障术后视力的主要原因,糖尿病患者白内障术后更容易发生PCO.研究证实,去整合素Echistatin（Ecs）能抑制体外晶状体上皮细胞（LECs）的增生,从而抑制PCO的形成,但其机制和安全剂量有待证实.目的 研究不同质量浓度Ecs对糖尿病兔早期PCO发生过程中LECs增生的抑制作用及Ecs在眼内应用的安全剂量.方法 用随机数字表法将15只新西兰大白兔分为5个组,用四氧嘧啶兔耳缘静脉注射法建立兔糖尿病模型,然后兔右眼行常规晶状体囊外摘出术,术后单纯糖尿病兔前房注入蒸馏水0.2 ml作为对照组,术后按照分组前房内分别注入5.0、7.5、10.0和15.0 μg/ml的Ecs0.2 ml.术后裂隙灯生物显微镜下观察眼前段炎症反应及PCO形成和分级情况.术后10d取出兔右眼,制备常规石蜡切片,采用免疫组织化学法检测PCO发生过程中LECs中增生细胞核抗原（PCNA）的表达以确定Ecs对PCO抑制的有效剂量;采用常规组织病理学方法检查角膜和视网膜结构的形态学改变,并采用TUNEL法检测角膜内皮细胞及视网膜内核层细胞的凋亡情况,以确定Ecs对眼内组织的安全剂量.结果 各组兔实验眼晶状体囊外摘出术后出现不同程度的角膜水肿及前房渗出,对照组和5.0 μg/ml Ecs组术后3～5 d炎症反应消失,≥7.5 μg/ml Ecs组术眼均于术后7d恢复正常.术后对照组和5.0 μg/ml Ecs组PCO均为1～2级,≥7.5 μg/ml Ecs组术眼PCO均为0级.对照组、5.0 μg/ml Ecs组、7.5 μg/ml Ecs组和10.0 μg/ml Ecs组LECs中PCNA阳性细胞表达值（A值）的总体比较差异有统计学意义（F=18.006,P=0.001）,与对照组比较,7.5和10.0 μg/ml Ecs组LECs中PCNA阳性细胞表达值明显下降,差异均有统计学意义（P=0.010、0.001）.各组兔眼角膜内皮细胞排列整齐,视网膜内界膜与各层组织结构完整.TUNEL法检测可见≤10.0 μg/ml Ecs各组与对照组间角膜内皮细胞及视网膜内核层?     

慢病毒介导的单纯疱疹病毒胸苷激酶基因/丙氧鸟苷体系抑制人晶状体上皮细胞的研究

目的探讨慢病毒介导的单纯疱疹病毒胸苷激酶基因／丙氧鸟苷体系（HSV—tk／GCV）对人晶状体上皮细胞（LECs）系的杀伤效应的机制。方法HSV—tk基因和增强型绿色荧光蛋白（EGFP）报告基因共表达的慢病毒感染细胞为试验组,仅表达EGFP的慢病毒感染细胞和正常细胞为对照组。流式细胞仪检测病毒的转染效率,荧光显微镜观察及基因组PCR和RT-PCR检测基因在LECs内表达,DNA ladder和电镜观察该系统对LECs的杀伤作用,检测HSV—tk／GCV系统的浓度时间依赖性和旁观者效应。结果HSV—tk整合入LECs并高效表达。10～25μg／ml的GCV能明显诱导转染HSV—tk的细胞凋亡或坏死,且随GCV浓度升高和作用时间延长效应增强,且存在明显的旁观者效应,而对照组细胞无显著改变。当GCV浓度〉25μg／ml,对照组细胞的增殖也被显著抑制。结论GCV可高效杀伤表达HSV-tk的LECs,且旁观者效应显著增强了HSV—tk／GCV系统的杀伤效果。HSV—tk和EGFP共表达的慢病毒载体能高效稳定转染LECs。     

A preliminary study on the prevention of posterior capsule opacification by photodynamic therapy with bacteriochlorin A in rabbits

PURPOSE: Photodynamic therapy (PDT) with bacteriochlorin a (BCA) has proven to be successful in the treatment of cancers and to be cytocidal for lens epithelial cells (LECs) in culture. The present study aimed to determine whether PDT with BCA is also effective in destroying LECs in the capsular bag in vivo and could therefore be a strategy for prevention of posterior capsule opacification (PCO). MATERIALS AND METHODS: BCA was obtained by saponification and acid hydrolysis of bacteriochlorophyll a and was formulated in 30% polyethylene glycol, 20% ethanol and 50% water. Nine albino rabbits were anesthesized and both pupils dilated. Extracapsular lens extraction by phacoemulsification was performed on both eyes. One eye of each animal served as control. In the other eye, 1.5 ml BCA (10 or 50 microg/ml) was injected in the capsular bag and after 10 min, the eye was illuminated with a diode laser (wavelength 760 nm) for 10 or 15 min. Six weeks after surgery, the rabbits were sacrificed and the globes were enucleated, the capsular bags and the corneas removed, fixed and examined using stereomicroscopy and light microscopy. RESULTS: In the control capsular bags, extensive proliferation of LECs and formation of a complete ring of Soemmering was found, while in the PDT-treated capsular bags, LEC proliferation was markedly diminished and an incomplete irregular and much thinner ring of Soemmering was formed. Using the presently described application, the corneas of the PDT-treated animals were opaque and swollen and had lost their endothelial lining. CONCLUSION: PDT with BCA induces cell death in LECs and greatly reduces the formation of a ring of Soemmering. Therefore, it could be a promising novel means of prevention of PCO, provided the total length of the treatment can be substantially reduced and the negative effects on corneal transparency avoided.     

Selenium functionalized intraocular lenses inhibit posterior capsule opacification in an ex vivo canine lens capsular bag assay

The purpose of this study was to determine the inhibitory effect of selenocystamine coated intraocular lenses (IOLs) on the formation of posterior capsule opacification (PCO) in an ex vivo canine lens capsular bag assay. Selenocystamine was covalently bound to the surface of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) discs. Three groups of canine lens capsules (6 coated IOLs (SeIOLs), 7 non-coated control IOLs and 8 empty capsules) were cultured for 10 days. During the culture period PCO was scored based on visual inspection of the capsules using phase contrast microscopy. On day 10 all the capsules were prepared for light microscopic examination and lens epithelial cells (LECs) were quantified. Proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA) and cleaved caspase-3 were examined by immunohistochemistry. Additionally, cell viability assays were performed on LECs cultured in tissue culture medium pre-incubated with either a SeIOL or control IOL. The viability assays demonstrated that no detectable cytotoxic leachables were associated with the functionalized IOLs. The central posterior capsule was free of cells underneath all SeIOLs, although large numbers of LECs populated the capsular periphery. Apoptotic cells were observed underneath the periphery of some SeIOLs. Both the PCO scores and LEC counts of SeIOL containing capsules were significantly lower than those of control group capsules (p < 0.01 and p = 0.0004, respectively).The use of selenium functionalized IOLs resulted in a significant reduction of PCO in this ex vivo model. Binding of selenocystamine to a foldable IOL may provide an effective method to prevent population of the central posterior capsule with LECs.     

Retrovirus-mediated transfer of a suicide gene into lens epithelial cells in vitro and in an experimental model of posterior capsule opacification.

PURPOSE. The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification and was found to be an important feature contributing to opacification of the posterior capsule. We investigated the feasibility of killing the residual lens epithelial cells by retroviral-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, a well-studied suicide gene, into rabbit lens epithelial cells followed by ganciclovir (GCV) treatment. METHODS. The capacity of retroviral vectors to transfer genes into rabbit lens epithelial cells was determined either in vitro (culture of rabbit lens epithelial cells) or in vivo (experimental model of PCO in rabbits) using cDNA encoding the beta-galactosidase (LacZ) reporter gene. To evaluate the efficiency of suicide gene therapy (infection with retroviral vectors encoding the HSV-tk gene followed by GCV treatment) we determined the sensitivity of HSV-tk infected lens epithelial cells to different concentrations of GCV in vitro. Then, in an experimental model of PCO, rabbits were treated with HSV-tk retroviral vectors at the end of the surgery and they received repeated intracameral and intravitreal injections of GCV at the concentration determined by the in vitro experiments. RESULTS. Infection efficiency using LacZ retroviral vectors was about 29% in vitro and 10% in vivo. After infection of the HSV-tk cDNA in vitro, the cell killing effect of GCV was evaluated. A significant enhancement (four- to five-fold) of the cell sensitivity to GCV was shown in FLY-DFGtk as compared with mock infected (P < 0.01) cells even without selection of the HSV-tk positive cells. The GCV concentration leading to 50% reduction in cell number (IC50) was 50 microg/ml. In vivo infection with a HSV-tk vector led to the tk gene transfer into lens epithelial cells. Despite this local HSV-tk gene expression, we could not prevent capsule opacification. CONCLUSIONS. Lens epithelial cells were successfully infected both in vitro and in vivo by beta-galactosidase and HSV-tk genes via retroviral vectors. In vitro infected lens epithelial cells displayed a strong sensitivity to GCV treatment. In vivo, we could not prevent capsule opacification in the rabbit model, very likely due to the limited level of the HSV-tk gene expression. However, our results suggest that virus-mediated suicide gene therapy might be a feasible treatment strategy to prevent capsule opacification with a more powerful vector.     

In vitro study on the closure of posterior capsulorrhexis in the human eye

PURPOSE: An unexplained clinical observation is the development of posterior capsular opacification (PCO), even when the central part of the posterior capsule has been removed. The purpose of this study was to investigate in vitro the mechanisms involved in the closure of the posterior capsulorrhexis in a capsular bag model. METHODS: A sham extracapsular cataract extraction was performed in 71 human donor eyes, followed by a central posterior capsulorrhexis 3 to 4 mm in diameter. Each capsular bag was pinned to a PMMA ring with a central hole of 5 mm and placed in a Petri dish. The capsular bags were cultured and monitored for 3 to 7 weeks by phase-contrast microscopy, after which they were prepared for light, transmission, and scanning electron microscopy. RESULTS: Proliferation of lens epithelial cells (LECs) within the posterior rhexis area was found in 22 cases (31%) of which 3 had a complete closure. In the absence of the posterior capsule, a monolayer of LECs was observed growing on a basal lamina, consisting of loosely arranged fibers. Further observations on noncultured capsular bags revealed that this basal lamina corresponds to the anterior hyaloid membrane. CONCLUSIONS: This study corroborates the clinical observation that LECs that remain after cataract extraction have the potential to proliferate, in the absence of their natural substrate, on a basal lamina of vitreous origin and are able to close the posterior capsulorrhexis partially or totally in approximately one third of cases.     

Prevention of posterior capsule opacification by intraoperative single-dose pharmacologic agents

PURPOSE: To determine whether an intraoperative single dose of dexamethasone, diclofenac, ethylenediaminetetraacetic acid (EDTA), a combination of EDTA and RGD peptide (arginine-glycin-aspartic acid sequence), or mitomycin-C (MMC) is a pharmacological means of preventing or reducing the development of posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, Celal Bayar University, School of Medicine, Manisa, and Department of Pathology, Dokur Eylül University, School of Medicine, Izmir, Turkey. METHODS: Fifty-four rabbits were randomly divided into 6 groups. Dexamethasone (4 mg/cc), diclofenac (2.5 mg/cc), EDTA (8 mg/cc), a combination of EDTA and RGD peptide (2.5 mg/cc), or MMC (0.04 mg/cc) was given, 0.1 cc by hydrodissection and 0.9 cc into the capsular bag after phacoemulsification. The sixth group served as a control group. After 3 months, the PCO was graded clinically and the proliferation of lens epithelial cells (LECs) was evaluated histologically. RESULTS: The drugs were significantly effective in preventing PCO compared with the control (P <.005). Dexamethasone had a weaker effect than the other drugs. In histological analysis, although monolayer LECs in the dexamethasone and diclofenac groups were observed, there was no proliferative activity on the posterior capsules in the EDTA, EDTA+RGD, and MMC groups in contrast to the multilayer cells in the control. CONCLUSIONS: Intraoperative single-dose application of EDTA, EDTA+RGD peptide combination, and MMC significantly prevented the development of PCO in rabbit eyes. Diclofenac was less effective but also reduced PCO. Although dexamethasone did not prevent the proliferation of LECs, it decreased PCO clinically.     

莱菔硫烷激发的细胞自噬潮对离体人晶状体囊上细胞增生的抑制作用

背景 研究已证实莱菔硫烷(SFN)为一种有效的化学预防剂,能调控生物体不同的分子机制以抑制细胞的无序生长.自噬是人体细胞维持自身内环境的生物过程,探讨SFN对晶状体上皮细胞(LECs)生物学行为的影响及其与细胞自噬的关系有助于为后发性白内障(PCO)的预防和靶向治疗提供新的思路.目的 探讨SFN诱导人LECs凋亡的直接作用及其激发细胞自噬潮的作用机制. 方法 将离体24 h内的人供体眼行常规白内障超声乳化手术,取出的完整晶状体囊袋置于含体积分数2％胎牛血清的EMEM中培养,建立PCO囊袋模型.按照分组分别于培养基中添加0(空白对照组)、1、10和100 μmol/L SFN培养30 d,观察各组晶状体囊袋上细胞的生长情况,并采用免疫荧光技术检测细胞中F-actin和Vimentin的表达.用含5％胎牛血清的EMEM培养晶状体上皮细胞系FHL124,将培养的细胞分为空白对照组1、10、30和100 μmol/L SFN处理组,行乳酸脱氢酶(LDH)释放试验以测定各组细胞LDH释放的百分率;采用划痕试验检测各组细胞划痕面积的变化以评估细胞迁移能力;透射电子显微镜下观察各组细胞中自噬囊泡的超微结构及数量;采用Western blot法检测SFN组、SFN+3-MA组和3-MA处理的细胞自噬活动特异蛋白LC3的相对表达. 结果 各不同质量浓度SFN组晶状体囊袋上细胞覆盖率总体比较差异有统计学意义(F=48.57,P＜0.01).10 μmol/L SFN组明显低于空白对照组和1μmol/L SFN组.免疫荧光检测技术表明空白对照组增生细胞中F-actin和Vimentin染色阳性,增生的细胞排列致密,随着SFN质量浓度升高,F-actin和Vimentin阳性细胞密度下降,100 μmol/L SFN组未发现F-actin和Vimentin阳性细胞.空白对照组及1、10、30和100 μmol/L SFN组细胞LDH释放量(A值)分别为0.19±0.03、0.39±0.06、0.56±0.07、0.68 ±0.08和0.89±0.09,其中10、30、100 μmol/L SFN组细胞LDH释放量均明显高于空白对照组,随着SFN质量浓度升高,细胞LDH释放量逐渐增加,均明显高于其相邻的低质量浓度组,差异均有统计学意义(均P＜0.01).随着SFN质量浓度的升高,划痕面积减少率呈下降趋势,10、30、100 μmol/L随着SFN质量浓度升高,划痕面积的减少均明显低于其相邻的低质量浓度组,差异均有统计学意义(均P＜0.05).空白对照组、SFN组、SFN+3-MA组和3-MA细胞中LC3-Ⅱ蛋白表达的灰度值分别为0.423±0.003、14.543±0.024、0.668±0.024和0.576±0.056,SFN+3-MA和3-MA组LC3-Ⅱ蛋白表达的灰度值均明显低于SFN组,差异均有统计学意义(均P＜0.01).空白对照组及1、10和100 μmol/L SFN组间自噬小体数量分别为4.07 ±0.32、4.13±0.34、9.21 ±0.53和21.02±1.34,其中10和100 μmol/L SFN组细胞中自噬小体数量均明显高于空白对照组及1μmol/L SFN组,差异均有统计学意义(均P＜0.01). 结论 SFN可以激发人LECs自噬潮,从而抑制离体人晶状体囊上LECs的增生,促进LECs的死亡.本研究结果有望成为预防和治疗PCO的新药.     

Lens epithelial cell proliferation in human posterior capsule opacification specimens

In previous in vitro studies on capsular bags it was shown that, after a sham extracapsular cataract extraction (ECCE) on human donor eyes, lens epithelial cells (LECs) show, in the short term, a dramatically elevated mitotic activity as compared to that in the intact lens. The long term in vivo proliferation of LECs in human lenses after ECCE and intraocular lens (IOL) implantation has not been studied until now. In the present study, the mitotic activity of LECs in human post-mortem eyes with posterior capsule opacification (PCO) was investigated. Human lenses with signs of PCO were dissected from donor eyes and incubated in MEM, supplemented with fetal calf serum, for 1 day (n = 10) or 7 days (n = 9). Six additional specimens were cultured for 7 days after removal of the IOL and lens fibres. After the incubation period, mitotic activity was estimated using the BrdU procedure and the Ki67 proliferating cell marker.The mean number of BrdU-positive nuclei in the intact PCO specimens was at a level of 7.5 (day 1) and 6.5 (day 7). Removal of the IOL and the lens fibres leads to a ten-fold increase in BrdU positive cells (mean = 84.5). No correlation with donor age was found. The Ki67 observations corroborate the BrdU results.The results demonstrate that after an initial rise in proliferative activity, as shown in the capsular bag model, the mitotic activity of LECs returns to a rate comparable to that in intact cultured non-cataractous lenses. As in control lenses, removal of lens fibres significantly elevated the proliferative activity of the remaining LECs. Suppression by newly formed differentiated lens fibres in the in vivo capsular bag may be responsible for this return to control levels of mitotic activity of LECs in the PCO specimens.     

核心蛋白多糖对兔晶状体上皮细胞增生的抑制作用

背景 研究发现,白内障囊外摘出术后组织修复反应可诱导房水中活性转化生长因子β(TGF-β)的增加,残留的晶状体上皮细胞(LECs)移行、分化,细胞外基质沉积,引起上皮-间质转化,导致后囊膜混浊(PCO)的发生.寻求有效的抑制LECs增生的药物对于临床上防治PCO的发生具有重要意义. 目的 探讨核心蛋白多糖( decorin)对兔LECs增生的抑制作用及其剂量-效应与时间-效应的关系. 方法 兔LECs细胞株进行培养和传代,将处于指数生长期的细胞以8×106个/L密度接种于96孔板,将0.1、1.0、10.0 mg/Ldecorin分别加入培养基中培养24、48、72 h,加入体积分数0.1％ DMSO培养的细胞为阳性对照组,常规培养基培养的细胞为空白对照组.MTT比色法分别测定不同质量浓度的decorin作用于LECs不同时间后的细胞生长抑制率;采用流式细胞技术测定各组细胞的细胞周期,用ELISA法检测各组培养液上清中TGF-β的水平,逆转录聚合酶链反应(RT-PCR)法测定TGF-βmRNA在LECs中的表达,免疫细胞化学法观察α-平滑肌肌动蛋白(α-SMA)的表达.结果 ELISA检测结果表明,各组培养基上清液中TGF-β表达量的差异有统计学意义(F=39.24,P=0.03),不同质量浓度组TGF-β水平均明显低于空白对照组,差异有统计学意义(P＜0.01),1.0 mg/L、10.0 mg/L decorin组TGF-β水平均明显低于0.1 mg/L decorin组(P＜0.05).MTT比色法结果显示,≥1.0 mg/L decorin组LECs增生的抑制率明显高于空白对照组,各质量浓度decorin组药物作用48 h和72 h后LECs增生的抑制率明显高于24 h的值,药物作用72 h后LECs增生的抑制率明显高于48 h的值,差异均有统计学意义(P＜0.05),各质量浓度decorin组G0/G1期LECs所占比例均较空白对照组明显增加,差异均有统计学意义(P＜0.05).RT-PCR检测显示,TGF-β mRNA的表达随着decorin质量浓度的升高而降低.免疫组织化学染色表明,10.0 mg/L decorin组α-SMA在LECs中的表达明显弱于空白对照组. 结论 Decorin可以抑制LECs的增生,也可以诱导LECs凋亡,其效应呈明显的剂量和时间依赖性,有望成为最具潜力的防治PCO的药物之一.