抗增生药物对人视网膜神经胶质细胞的抑制作用

目的寻找抑制视网膜、玻璃体内细胞增生的有效药物;明确抗增生药物秋水仙碱、道诺霉素和５－氟尿嘧啶（５－Ｆｕ）对体外培养的人视网膜胶质（ｒｅｔｉｎａｌ ｇｌｉａ,ＲＧ）细胞的作用。方法用 ＭＴＴ法测定秋水仙碱（０．５～１６．０μｇ／ｍｌ）、道诺霉素（０．１～３．２μｇ／ｍｌ）和５－Ｆｕ（０．５～１６．０μｇ／ｍｌ）对体外培养人ＲＧ细胞的作用。结果秋水仙碱（１．０～１６．０μｇ／ｍｌ）、道诺霉素（０．２～３．２０μｇ／ｍｌ）和 ５－Ｆｕ（１．０～１６．０μｇ／ｍｌ）３组药物对培养细胞均有抑制作用,与对照组均差别显著（Ｐ＜０．０１）,ＩＤ_（５０）分别为３．１１μｇ／ｍｌ,０．７９μｇ／ｍｌ和５．２３μｇ／ｍｌ。结论秋水仙碱、道诺霉素和５－Ｆｕ对ＲＧ细胞有明显抑制作用。     

MTT比色法测定药物对晶体上皮细胞增殖的影响

目的筛选防治后囊混浊的药物。方法进行人胚胎晶体上皮细胞（ｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌｓ，ＬＥＣ）的培养并用ＭＴＴ比色法检测了肝素、氟脲嘧啶、双氯灭痛、顺铂对人胚胎晶体上皮细胞增殖的影响。结果肝素１００～２５００Ｕ／ｍｌ，氟脲嘧啶０．１～１００μｇ／ｍｌ，顺铂１０～５００μｇ／ｍｌ作用２４ｈ和７２ｈ对胚胎晶体上皮细胞增殖有明显抑制作用（Ｐ＜０．０５）。作用７２ｈ抑制率高于２４ｈ（Ｐ＜０．０５）。双氯灭痛和对照组相比无明显差异（Ｐ＞０．０５）。结论肝素、氟脲嘧啶、顺铂均为剂量依赖型和时间依赖型药物，均能有效抑制晶体上皮细胞的生长；双氯灭痛对晶体上皮细胞增殖无明显抑制作用。     

r—干扰素和地塞米松抑制成纤维细胞的实验研究

成纤维细胞的增殖与许多眼病有关，作者采用ＭＴＴ比色法检测，ｒ－干扰素和地塞米松对成纤维细胞的抑制作用。结果表明，ｒ－干扰素在０．１－１００００μ／ｍｌ剂量时对成纤维细胞增殖没有直接抑制作用，地塞米松在０．０１－１０００μｇ／ｍｌ剂量对成纤维细胞有明显抑制作用。     

r－干扰素和地塞米松抑制成纤维细胞的实验研究

成纤维细胞的增殖与许多眼病有关，作者采用ＭＴＴ比色法检测，ｒ－干扰素和地塞米松对成纤维细胞的抑制作用。结果表明，ｒ－干扰素在０．１─１００００μ／ｍｌ剂量时对成纤维细胞增殖没有直接抑制作用，地塞米松在０．０１─１０００μｇ／ｍｌ剂量时对成纤维细胞有明显抑制作用。     

增殖性糖尿病视网膜病变患者玻璃体中血管内皮细胞生长因子含量的检测

目的检测增殖性糖尿病视网膜病变（ｐｒｏｌｉｆｅｒａｔｉｖｅｄｉａｂｅｔｉｃｒｅｔｉｎｏｐａｔｈｙ，ＰＤＲ）患者玻璃体中血管内皮细胞生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ，ＶＥＧＦ）的含量并与正常人进行对比研究，探讨ＶＥＧＦ在ＰＤＲ病理过程中的作用。方法应用酶联免疫吸附测定法（ｅｎｚｙｍｅｌｉｎｋｅｄｉｍｍｕｎｏｓｏｒｂｅｎｔａｓｓａｙ，ＥＬＩＳＡ）对７例正常人及１９例ＰＤＲ患者玻璃体中的ＶＥＧＦ进行定量分析研究。结果７例正常人玻璃体中，ＶＥＧＦ的含量为０．１８～０．６０ｎｇ／ｍｌ，平均值为０．３５ｎｇ／ｍｌ。１９例ＰＤＲ患者玻璃体中ＶＥＧＦ的含量升高，范围为０．８４～１５．６４ｎｇ／ｍｌ，平均值为５．６６ｎｇ／ｍｌ。两组比较，差异有显著性（Ｐ＜０．０５），其中男性组玻璃体中ＶＥＧＦ的含量为０．８４～１５．６４ｎｇ／ｍｌ，平均值为４．８３ｎｇ／ｍｌ；女性组为１．３４～１２．３４ｎｇ／ｍｌ，平均值为７．０８ｎｇ／ｍｌ，两组比较，差异无显著性（Ｐ＞０．０５）。结论ＰＤＲ患者玻璃体中ＶＥＧＦ的含量升高，在ＰＤＲ的病理过程中起一定作用     

防治增殖性玻璃体视网膜病变的研究──榄香烯联合地塞米松对成纤维细胞抑制作用的实验研究

本文对兔皮肤成纤维细胞进行了体外培养，应用噻唑兰比色法（ＭＴＴ法）对榄香烯和地塞米松进行药物筛选，结果发现：榄香烯对培养的成纤维细胞的增殖具有明显的抑制作用，并使细胞形态发生显著变化，其５０％抑制率浓度（ＩＤ５０）为０．００８ｍｇ／ｍｌ；地塞米松对该细胞增殖的作用呈双重性，低浓度刺激细胞增生，高浓度抑制细胞增殖，其５０％抑制率浓度为１６８ｍｇ／ｍｌ。此两种药物５０％抑制率浓度联合应用，对培养的成纤维细胞增殖的抑制作用（抑制率：６２．８％）较单用榄香烯（抑制率：５２．４％，Ｐ＜０．０１）或单用地塞米松（抑制率：５５．２％ｐ＜０．０１）明显增强。并对此两种药物抗增殖的作用机理进行初步探讨。     

阿霉素,5—Fu,三尖杉醌碱及去炎松对体外培养的人视网膜色素上皮细胞…

应用＾３Ｈ－胸腺嘧啶核苷掺入及液体闪烁测量技术，观察不同浓度的阿霉素，５－氟尿嘧啶，三尖杉酯碱及去炎松对体外培养的人视网膜色素上皮细胞增殖的抑制作用。结果；阿霉素在２－３２ｎｍ／ｍｌ时，对ＲＰＥ细胞抑制率为８．５－７７．２＾，ＩＤ５０为１２ｎｇ／ｍｌ；５－Ｆｕ在０．２－３．２μｇ／ｍｌ时，对ＲＰＥ细胞抑制率为１４．５－８１．７％，ＩＤ５０为０．６８μｇ／ｍｌ；三尖杉酯碱在２．５－４０ｎｇ／ｍｌ，抑     

非瑟酮对人晶状体上皮细胞增殖和凋亡的影响

目的　研究非瑟酮（ｆｉｓｅｔｉｎ,Ｆｉｓ）在生理状态下及氧化应激状态下对人晶状体上皮细胞（ｈｕｍａｎｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌｌ,ＨＬＥＣ）增殖和凋亡的影响。方法　体外培养ＨＬＥＣ,通过Ｈ２Ｏ２氧化损伤建立氧化应激模型,设置空白对照组、Ｈ２Ｏ２组、Ｆｉｓ组和Ｆｉｓ＋Ｈ２Ｏ２组,其中Ｆｉｓ组和Ｆｉｓ＋Ｈ２Ｏ２组按Ｆｉｓ浓度（５μｇ?ｍＬ－１、１０μｇ?ｍＬ－１和２０μｇ?ｍＬ－１）分为３个亚组。分别于培养１２ｈ及２４ｈ后,倒置相差显微镜下观察各组细胞的形态学改变,运用ＭＴＴ法检测细胞增殖的变化,运用流式细胞技术检测细胞凋亡率的变化。结果　与空白对照组比较,Ｈ２Ｏ２组较多细胞出现典型的形态学改变,细胞增殖能力明显降低（１２ｈ、２４ｈ后分别为０．１１７６±０．０１５０和０．１１７２±０．００６１）,凋亡率明显增加（２４ｈ后为１２．３５％ ±１．２３％）,差异均有统计学意义（均为Ｐ＜０．０１）。不同浓度Ｆｉｓ组间的细胞在培养１２ｈ及２４ｈ后细胞形态均无明显改变,细胞增殖也无明显变化（Ｐ＞００５）。培养１２及２４ｈ后,与Ｈ２Ｏ２组比较,Ｆｉｓ＋Ｈ２Ｏ２组发生形态改变的细胞减少,细胞增殖能力明显改善,且随时间、Ｆｉｓ浓度增加其作用更明显（最高为０．３９９４±０．０２５７）（Ｐ＜０．０５）。培养２４ｈ后,与Ｈ２Ｏ２组凋亡率比较,不同浓度Ｆｉｓ＋Ｈ２Ｏ２组的细胞凋亡率逐渐降低,依次为（９．９９±１．５３）％、（５．８０±１．５５）％、（３．５８±０．７３）％,差异有统计学意义（Ｐ＜０．０５）。结论　一定浓度的Ｆｉｓ在一定时段对生理状态下的ＨＬＥＣ增殖无明显影响。在氧化应激状态下,Ｆｉｓ呈时间和浓度依赖性地改善ＨＬＥＣ增殖能力,呈浓度依赖性地降低ＨＬＥＣ凋亡率。     

表皮生长因子对晶状体和角膜上皮细胞增生的刺激作用

目的探讨不同浓度的表皮生长因子（ＥＧＦ）对晶状体和角膜上皮细胞增生的刺激作用。方法利用培养的兔晶状体和角膜上皮细胞，通过ＭＴＴ方法，检测细胞增殖密度。培养晶状体上皮细胞的ＥＧＦ浓度（１～２５０ｎｇ／ｍｌ）分为８组；而培养角膜上皮细胞的ＥＧＦ浓度（４～１０００ｎｇ／ｍｌ）分为１０组。结果ＥＧＦ浓度在３２ｎｇ／ｍｌ时，促晶状体上皮细胞的增生作用最强，与无血清组比较差异非常显著（Ｐ＜０．０１）。促角膜上皮细胞增生的最佳浓度为１６ｎｇ／ｍｌ，与无血清组比较差异显著（Ｐ＜０．０５）。结论上述结果为今后在细胞和分子水平上研究白内障和角膜伤口愈合的发生规律及其机制提供实验资料。     

肿瘤坏死因子α和表皮生长因子对晶状体上皮细胞增殖的作用

目的研究肿瘤坏死因子α（ＴＮＦα）和表皮生长因子（ＥＧＦ）对人、牛晶状体上皮细胞增殖的影响。方法采用ＭＴＴ比色法检测ＴＮＦα和ＥＧＦ对晶状体上皮细胞（ｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌ，ＬＥＣ）增殖的作用。结果０．１Ｕ／ｍｌ的ＴＮＦα即可明显促进人、牛晶状体上皮细胞的增殖；ＥＧＦ为１、１０ｎｇ／ｍｌ时可明显促进牛晶状体上皮细胞增殖。结论细胞因子ＴＮＦα、ＥＧＦ通过促进ＬＥＣ的增殖参与后囊混浊的形成     

Effect of heparin on proliferation of cultivated bovine lens epithelial cells]

The treatments proposed to date for the prevention of secondary cataract have shown limited efficacy or have not been satisfactory due to ocular toxicity. Since it has been demonstrated that heparin can inhibit the proliferative activity of smooth muscle cells and fibroblasts in vitro and in vivo, we examined the effect of heparin at concentrations ranging from 20 to 200 micrograms/ml on the proliferation of cultured bovine lens epithelial cells (BLEC) under various culture conditions: (1) serum-free medium (SFM); (2) SFM + aqueous humor 1:1; (3) SFM +1 and 10% fetal calf serum; (4) SFM +1% retinal extract; (5) SFM +50 micrograms/ml endothelial cell growth factor; (6) SFM +10 ng/ml epidermal growth factor; (7) SFM +10 ng/ml basic fibroblast growth factor. Heparin caused no cytotoxic effects in any of the experiments. With medium 2 and 3, heparin caused dose-dependent inhibition of cell proliferation at concentrations ranging from 10 to 50 micrograms/ml. Cells cultivated in medium 4-7 with the addition of 50 micrograms/ml heparin revealed increased proliferative activity when compared with the corresponding controls. The antiproliferative activity on BLEC in medium containing aqueous humor suggests that heparin is a valuable tool for the prevention of secondary cataract in vivo.     

拉坦前列素滴眼液对人眼球筋膜囊成纤维细胞增殖的影响

目的研究拉坦前列素滴眼液对体外培养的人眼球筋膜囊成纤维细胞的增殖有无促进作用。方法体外培养人眼球筋膜囊成纤维细胞。按1∶10比例逐级稀释拉坦前列素滴眼液(0.005%),使药物浓度依次为5.00、0.50、0.05μg/ml直至5×10-8μg/ml。采用噻唑蓝比色法检测在不同浓度药物作用下的细胞增殖情况,并在光学显微镜下观察细胞的形态变化。结果较高浓度组(5.00μg/ml)拉坦前列素滴眼液对人眼球筋膜囊成纤维细胞存在明显的杀伤作用,细胞几乎全部死亡,吸光度(A)值与对照组相比明显下降(P<0.01);低浓度组(<0.05μg/ml)A值较对照组无明显变化(P>0.05)。比较拉坦前列素滴眼液在不同作用时间下的剂量效应曲线,24、48及72h差异均无统计学意义(P>0.05)。结论拉坦前列素滴眼液不能促进体外培养的人眼球筋膜囊成纤维细胞的增殖,在较高浓度时具有细胞毒性,在较低浓度时则对细胞增殖无明显影响。     

Bifunctional Effect of Human HFN—γon Cultured Human Fibroblasts from Tenon‘s Capsule

PURPOSE: To study the effect of human IFN-gamma on in vitro cultured human fibroblasts from Tenon's capsule. MATERIALS AND METHODS: The effect of different concentrations of human IFN-gamma and mitomycin-C (MMC), 5-fluorouracil (5-Fu) on cultured human Tenon's capsule fibroblasts (HTCF) was measured using a MTT [3-(4, 5-dimethylthiazo-2-yl)]-2, 5-diphenyltetrazolium bromide; Thiazolyl blue) colorimetric assay. The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9.0 version. The difference was considered to be significant if P < 0.05. RESULTS: The effects of MMC and 5-Fu on the growth of HTCF were negative, while the effects of IFN-gamma on the growth of HTCF were both negative (10(2)-10(4) units/ml in two experiments) and positive (10(6), 10(5), 10 units/ml in two experiments). The inhibition rate of MMC ranged from 5.73% to 46.9%, which was similar to the inhibition rate of 5-Fu ranged from 12.49% to 38.92% (P = 0.351). The inhibition rate of IFN-gamma in two experiments was smaller than MMC and 5-Fu (P < 0.05). CONCLUSION: IFN-gamma has bifunctional effect (both enhancement and inhibition) on proliferation of cultured HTCF. The antiproliferative effect of IFN-gamma was weaker than MMC and 5-Fu. Further study has to be carried out to document the inhibition of scar formation of filtration bleb by IFN-gamma and the molecular mechanisms of its bifunctional effect on HTCF proliferation.     

晶状体和玻璃体提取物及地塞米松影响Tenon囊成纤维细胞增殖和胶原合成的实验研究

目的　探讨晶状体和玻璃体提取物对猪眼筋膜囊(Tenon囊)成纤维细胞增殖和胶原合成的影响及其在糖皮质激素作用下的变化。方法　体外培养猪眼Tenon囊成纤维细胞,应用噻唑蓝(MTT)比色法和逆转录聚合酶链反应(RT -PCR)法观察在晶状体和玻璃体提取物及地塞米松单独或联合作用下,Tenon囊成纤维细胞增殖和胶原合成的变化。结果　与对照细胞吸光度(A值)(0 .305±0.013)比较, 10mg/L晶状体提取物( 0. 411±0. 000 )和10mg/L玻璃体提取物( 0. 349±0 .027)作用2d即具有促进细胞生长作用,差异有统计学意义(P=0. 00);且随着晶状体提取物、玻璃体提取物浓度的增高,A值相应增加,差异均有统计学意义(P=0. 00)。Ⅰ型胶原的相对含量在晶状体提取物作用细胞内为12 290±231,在玻璃体提取物作用细胞内为10 .853±231,与对照细胞(9389±178)比较,差异均有统计学意义(P<0. 01)。地塞米松对125mg/L晶状体提取物和125mg/L玻璃体提取物作用的Tenon囊成纤维细胞增殖分别具有一定抑制作用,差异有统计学意义(P=0. 00)。地塞米松和提取物联合作用细胞内Ⅰ型胶原的相对含量与提取物单独作用细胞比较,差异均无统计学意义(P>0 .05)。结论　晶状体和玻璃体提取物均可明显刺激Tenon囊成纤维细胞增殖,促进Tenon囊成纤维细胞内胶原合成,     

The long-term effects of 5-fluorouracil and sodium butyrate on human Tenon's fibroblasts.

The use of subconjunctival 5-fluorouracil (5-FU) in the first weeks after filtration surgery may ensure long-term bleb survival despite a continuing proliferative stimulus such as in eyes with neovascular glaucoma. In addition, long-term side effects may occur, such as increasing bleb thinning. To ascertain the long-term effects of 5-FU and sodium butyrate, an agent with differentiating and antiproliferative properties, we exposed proliferating human Tenon's capsule fibroblasts to different concentrations of the drugs. The cells were exposed to 5-FU for 1-12 d. The cells were subsequently observed for up to 30 d. Cell proliferation was assessed using cell counting and bromodeoxyuridine uptake, and cell viability was assessed with trypan blue uptake. 5-FU and sodium butyrate inhibited fibroblast proliferation during the treatment period. Higher concentrations of 5-FU (100 and 1000 micrograms/ml) for as little as 1 d resulted in no significant increase in the number of fibroblasts for at least 29 d after treatment was stopped, despite continued stimulation with serum. When treatment with sodium butyrate was stopped, there was greater recovery of proliferation. At a constant concentration of 1000 micrograms/ml of 5-FU for 3 or more days, or a concentration of 100 mmol/l sodium butyrate for 12 d, the entire fibroblast population gradually died over the 30 d period. Thus, short-term treatment with 5-FU may result in long-term inhibition of proliferation of fibroblasts. Long-term inhibition depends on the duration of treatment or on the concentration of 5-FU. Short-term treatment may be affecting the ability of the tissues at the bleb site to heal in the long term. Different dosage regimens may have advantages and are discussed.     

Bifunctional Effect of Human IFN-γ on Cultured Human Fibroblasts from Tenon's Capsule

Purpose: To study the effect of human IFN-r on in vitro cultured human fibroblasts from Tenon's capsuleMaterials and methods: The effect of different concentrations of human IFN-r and mitomycin-C (MMC), 5-fluorouracil (5-Fu) on cultured human Tenon's capsule fibroblasts (HTCF) was measured using a MTT [3-(4, 5-dimethylthiazo-2-yl)] -2, 5-diphenyltetrazolium bromide; Thiazolyl blue) colorimetric assay. The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9. 0 version. The difference was considered to be significant if P < 0. 05. Results: The effects of MMC and 5-Fu on the growth of HTCF were negative, while the effects of IFN-r on tne growth of HTCF were both negative (102 - 104 units/ml in two experiments) and positive (106, 105, 10 units/ml in two experiments) . The inhibition rate of MMC ranged from 5. 73% to 46. 9%, which was similar to the inhibition rate of 5-Fu ranged from 12. 49% to 38. 92% ( P = 0. 351) . The inhibition rate of IFN-'Y in two experiments was     

Growth of human corneal endothelial cells in culture

We investigated the effects of various culture conditions on the growth of normal human corneal endothelial cells in culture. Falcon Primaria tissue culture plastic was found to provide a more suitable surface for endothelial cell growth than the conventional Corning tissue culture plastic. Also, media containing 10% fetal bovine serum and 5% calf serum (complete media) facilitated the growth of human cells better than those containing Nu-serum. Supplementation with epidermal or fibroblast growth factor (10 and 100 ng/ml) to the complete media had no effect on human endothelial cell growth. Chondroitin sulfate at low concentrations (100 micrograms/ml to 1 mg/ml) also showed little effect. At high concentrations (13.5 and 25 mg/ml), however, chondroitin sulfate significantly promoted human corneal endothelial cell growth during a 1- to 2-week incubation period. From the 37 cultures initiated, outgrowth from explants appeared within 3 to 7 days. Cells were polygonal in shape and, at confluency, formed a continuous monolayer. We attained a success rate of 87% (7/8) growing primary cultures from donors under 20 years of age and a 59% (17/29) success rate from older donors.     

The effect of 5-fluorouracil and cytarabine on human fibroblasts from Tenon's capsule

The in vitro cellular inhibitory effects of two pyrimidine antimetabolites, 5-fluorouracil (5-FU) and cytarabine (ara-C), on the attachment and proliferation of human Tenon's capsule fibroblasts after 1, 2, 3, 5, 7, and 10 days of growth were measured with a Coulter counter, a colorimetric method using the endogenous enzyme hexosaminidase, and 3H-thymidine uptake. Neither 5-FU nor ara-C affected cell attachment. The 50% inhibitory dose (ID50) for 5-FU, as measured by the Coulter counter and hexosaminidase assay, was 0.2 and 0.4 micrograms/ml, respectively, at day 5 and decreased to 0.01 and 0.10 micrograms/ml, respectively, on later days. The ID50 for ara-C as measured by the Coulter counter and hexosaminidase assay was 0.01 and 0.1 micrograms/ml at day 3 and remained constant over time. Much lower ID50s were measured by thymidine uptake for both drugs. These findings may indicate that 5-FU has a delayed effect on cellular proliferation due to conversion into more active metabolites. The ara-C has a direct and constant inhibitory effect on cellular proliferation and is ten times more potent than 5-FU as an antiproliferative drug. Thus ara-C may have clinical utility in preventing failure of glaucoma filtering surgery.     

Ciprofloxacin and dexamethasone inhibit the proliferation of human retinal pigment epithelial cells in culture.

We investigated the inhibition of proliferation of human retinal pigment epithelial cells in vitro by the 4-quinolone, ciprofloxacin, and the steroid, dexamethasone. The concentration of ciprofloxacin that inhibited growth by 50% (IC50) was found to be 14.1 micrograms/mL. Growth was 100% inhibited at 83 micrograms/mL. At 166 micrograms/mL, all the cells became completely detached and appeared dead at the end of seven days. The IC50 for dexamethasone in RPE cells was found to be 141 micrograms/mL. A dexamethasone concentration of 1.3 mg/mL inhibited proliferation 100% after five days. When the two drugs were combined, the inhibitory effect was found to be additive; i.e., the IC50 dose of the two drugs in combination inhibited RPE cell proliferation by 75%. A combination of the two drugs was also tested for retinal toxicity in rabbit eyes. An examination of histological sections and electroretinograms showed that a dose of 100 micrograms of ciprofloxacin, alone or in combination with 200 micrograms of dexamethasone in saline, was not toxic to the rabbit retina. These studies indicate that a combination of ciprofloxacin and dexamethasone has the potential for reducing the risk of PVR formation and aiding in the prevention of endophthalmitis.     

Treatment of alkali-injured rabbit corneas with a synthetic inhibitor of matrix metalloproteinases.

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.