结缔组织生长因子在体外人视网膜色素上皮 细胞损伤模型中的表达和促移行作用

目的观察结缔组织生长因子（CTGF）在视网膜色素上皮（RPE）细胞损伤模型中的表达及其对RPE细胞移行的影响。方法利用体外培养的单层近融合期人RPE细胞，采用棉签和角膜移植用环钻做圆形细胞刮伤区，建立体外RPE细胞损伤模型。链霉亲和素-生物素复合物（SABC）免疫组织化学和原位杂交（ISH）方法检测RPE细胞受创后不同时间CTGF的表达及其转录水平mRNA的变化。并计数进入缺损区的RPE细胞数，定量观察CTGF对RPE细胞移行的影响以及地塞米松对CTGF这一作用的影响。结果免疫组织化学和原位杂交结果显示,创伤后6 h，创伤边缘RPE细胞表达CTGF呈弱阳性反应; 随着创伤后时间延长,阳性信号逐渐增强；伤后24、48 h创伤边缘移行的RPE细胞呈CTGF强阳性表达。重组人CTGF因子（rhCTGF）呈剂量依赖性刺激RPE细胞移行，地塞米松对CTGF诱导的RPE细胞移行作用有显著抑制作用。结论CTGF参与了RPE细胞创伤修复过程，这在增生性玻璃体视网膜病变等眼内增生性疾病中可能有重要意义。(中华眼底病杂志,2005,21:306-309)     

几丁糖抑制兔视网膜色素上皮细胞增殖作用的研究

增生性玻璃体视网膜病变的发生在很大程度上与视网膜色素上皮(retinal pigment epithelial,RPE)细胞的游走、移行、化生为类成纤维细胞且大量增殖有关[1-2].     

过度光照后诱导型一氧化氮合酶在大鼠视网膜色素上皮的表达

目的探讨过度光照诱导大鼠视网膜色素上皮（retinal pigment epithelium．RPE）细胞表达诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）的作用及其病理学意义。方法应用免疫组织化学的方法分别检测处于自然光照环境中的正常大鼠及接受强光过度照射后大鼠的视网膜RPE细胞中iNOS的表达情况。结果正常大鼠RPE细胞中未见iNOS的表达，而过度光照后大鼠RPE细胞的胞浆中表达高水平的iNOS，同时视网膜外核层感光细胞发生结构损伤。结论过度光照可诱导RPE细胞表达iNOS．这一异常改变可能是视网膜感光细胞结构损伤的重要原因。     

视网膜色素上皮细胞损伤机制研究进展

近年来许多研究表明,视网膜色素上皮(retinal pigment epithelium,RPE)细胞的损伤或增殖与多种视网膜疾病的发生发展有关。本文通过对RPE细胞损伤现状的阐述、以及它在视网膜病变过程中所起到的作用,期望从RPE细胞损伤机制寻找预防和治疗这些疾病的新途径。     

水蛭提取液诱导视网膜色素上皮细胞转分化的初步研究

药物诱导细胞分化是细胞分子生物学领域研究的热点课题，从中药中寻找和提取细胞的诱导分化剂是此类研究的新途径。本研究观察水蛭对视网膜色素上皮（retinal pigment epithelium，RPE）细胞转分化的影响。     

褪黑素对高糖刺激人视网膜色素上皮细胞诱导型一氧化氮合酶表达的影响

目的研究褪黑素对高糖刺激体外培养的人视网膜色素上皮（retinal pigment epithelial,RPE）细胞诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）表达的影响。方法培养人RPE细胞,分为对照组、甘露醇组、高糖组和高糖＋褪黑素组,作用48h后,光镜观察细胞形态;MTT法检测细胞活性;免疫荧光染色法和Western blot检测RPE细胞中iNOS的蛋白表达。结果MTT检测结果表明高糖可以抑制RPE细胞增生,加入褪黑素后,抑制作用减弱;免疫荧光染色法和Western blot检测结果表明,与对照组相比,高糖组iNOS蛋白表达明显增高,加入褪黑素后,iNOS表达被显著抑制。结论高糖可以抑制RPE细胞增生,诱导RPE细胞iNOS的表达上调,而褪黑素可减轻细胞损伤。     

氧化损伤对体外培养人视网膜色素上皮细胞PEDF的影响

目的研究氧化损伤对人视网膜色素上皮(retinal pigment epithelial,RPE)细胞表达色素上皮细胞衍生因子(pigment epithelium derived factor,PEDF)的影响。方法体外培养人RPE细胞,加入浓度为600μmol.L-1H2O2分别作用不同时间(2h、8h、24h),采用免疫细胞化学法检测PEDF蛋白的表达,逆转录聚合酶链式反应(RT-PCR)法检测PEDF mRNA。结果免疫细胞化学染色对照组即有PEDF的阳性表达,而氧化损伤2h、8h、24h PEDF蛋白阳性表达量逐渐减少,染色由棕黄色变为黄色。各时间段两组结果比较,差异均具有统计学意义(P<0.05)。RT-PCR检测PEDF mRNA量与PEDF蛋白表达变化一致。结论氧化损伤能促使人RPE细胞PEDF表达下调,并在一定范围内与作用时间有关。     

视网膜色素上皮损伤后相邻感光细胞及脉络膜血管的病理变化

目的:研究视网膜色素上皮(retinal pigment epithelium,RPE)损伤后相邻感光细胞和脉络膜血管的病理变化。方法:机械性刷除青紫蓝兔眼底髓线上方的RPE细胞,观察术后7,14,30d和90d清除区相邻感光细胞和脉络膜血管变化。结果:RPE细胞清除术后相邻的感光细胞发生退行性改变和死亡,脉络膜毛细血管萎缩。结论:RPE清除术可模拟萎缩型年龄相关性黄斑变性(age-related macular degeneration,AMD)及渗出型AMD剥膜以后的病理变化,为自体RPE移植挽救损伤的感光细胞的实验提供了良好的动物模型。     

视网膜色素上皮细胞体外凋亡过程中钙离子平衡与caspase-3基因表达

目的:研究钙通道拮抗剂-维拉帕米(verapamil,Ver)诱导视网膜色素上皮(retinal pigment epithelium,RPE)细胞凋亡过程中钙离子及凋亡基因caspase-3变化。方法:应用80mg/L的Ver分别作用健康人眼RPE细胞12,24及48h诱导凋亡,设立对照组。逆转录聚合酶链反应(RT-PCR)检测凋亡基因caspase-3的表达,采用Fluo-3/AM负载技术,MetaFluo4.5/coolsnapfx/IX70细胞内钙离子荧光成像系统测定每组20个RPE细胞钙荧光值,并计算RPE细胞内钙浓度([Ca2+]i)。结果:对照组RPE细胞Ca2+荧光分布胞核最强,胞质次之。Ver作用12,24及48h后,细胞内[Ca2+]i明显降低(P<0.01)。对照组RPE细胞可见caspase-3的mRNA有少量的表达。Ver作用12h后,可见caspase-3的mRNA有较高的表达,与对照组比较,具有显著性差异(P<0.01)。随着Ver作用时间的延长,caspase-3的mRNA表达逐渐增强,在48h时有所下降。结论:Caspase-3基因表达上调及RPE细胞内钙离子稳态失调可能在Ver诱导RPE细胞凋亡中起关键作用。     

氧化应激和自噬在干性年龄相关性黄斑变性发病中的作用

年龄相关性黄斑变性(age-related macular degeneration, AMD)是导致60岁以上人群视力障碍主要原因之一。干性AMD的主要病理特征是视网膜色素上皮(retinal pigment epithelium, RPE)细胞的退化死亡,而氧化应激是引起RPE损伤的主要因素。在RPE抵抗氧化应激损...     

Positive feedback regulation between MMP-9 and VEGF in human RPE cells

PURPOSE: The proteolytic activity of matrix metalloproteinases (MMPs) is involved in pathologic angiogenesis in the eye. However, it is unknown whether MMPs may stimulate the production of the major angiogenic factor, vascular endothelial growth factor (VEGF). The authors investigated whether MMP-2 and MMP-9 alter the expression of VEGF by retinal pigment epithelial (RPE) cells. They also sought to determine the effects of MMPs on cellular proliferation and migration and the effect of triamcinolone acetonide on MMP-9-evoked cellular responses. METHODS: Human RPE cell cultures were stimulated with MMP-2 or MMP-9. The gene expression and secretion of MMP-9 and VEGF were determined by real-time RT-PCR and ELISA, respectively. Cellular proliferation was investigated with a bromodeoxyuridine immunoassay, and chemotaxis was examined with a Boyden chamber assay. RESULTS: Under control conditions, RPE cells in vitro expressed a significantly higher amount of mRNA for MMP-2 than for MMP-9. Chemical hypoxia caused upregulation of the gene expression of both MMPs, whereas VEGF increased the gene expression and secretion of MMP-9. The hypoxic expression of MMP-9 was mediated by autocrine VEGF signaling. Exogenous MMP-9 increased the gene expression and secretion of VEGF, whereas MMP-2 reduced the secretion of VEGF. MMP-2 and MMP-9 did not alter the proliferation but stimulated the migration of RPE cells. Triamcinolone fully inhibited the stimulatory effect of MMP-9 on the expression of VEGF and the VEGF-evoked increase in the expression of MMP-9. However, triamcinolone had no effect on the motogenic effect of MMP-9. CONCLUSIONS: There is a positive feedback regulation between MMP-9 and VEGF in RPE cells. The hypoxic expression of MMP-9 may stimulate the production and secretion of VEGF under pathologic conditions. Triamcinolone inhibits the positive feedback regulation between MMP-9 and VEGF under hypoxic conditions through inhibition of the gene expression of MMP-9 and the secretion of VEGF.     

Modulation of matrix metalloproteinase and TIMP-1 expression by cytokines in human RPE cells

PURPOSE: The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is crucial for homeostasis of ocular extracellular matrices. To assess altered MMP activity as a determinant in the migration of human retinal pigment epithelial (RPE) cells, expression characteristics of several MMPs and TIMP-1 in RPE cell cultures were investigated. METHODS: Expression studies were performed with RT-PCR, ELISA, and immunofluorescence analysis. Secretion of MMP-2 was demonstrated by zymography. Migration of cytokine-stimulated RPE cells was evaluated with microporous membranes of permeable chambers. RESULTS: MMP-1, -2, -3, and -9; MT2-MMP; and TIMP-1 were expressed in cultured RPE cells. MMP-2 was detected on the cell surface and in secreted inactive and active forms. TGF-beta(2), IL-1beta, and TNF-alpha enhanced secretion of MMP-1, -2, and -3. TGF-beta(2) also stimulated MT2-MMP cell surface expression and release of TIMP-1. The mRNA levels of MMP-1, -2, and -3 and TIMP-1 were markedly increased by TNF-alpha and TGF-beta(2). MMP-2 mRNA levels were also upregulated by PDGF-BB. Migration of RPE cells stimulated by TGF-beta(2) or PDGF-BB was inhibited in presence of a synthetic MMP inhibitor. CONCLUSIONS: Proinflammatory cytokines and TGF-beta(2) play an important role in the upregulation of expression of MMP-1, -2, and -3 in RPE cells and account for a directional shift in the balance between MMPs and TIMPs. Facilitation of RPE cell migration stimulated by cytokines (i.e., TGF-beta(2) or PDGF-BB) in ocular diseases may be due to increased release of MMPs, in the presence of comparatively lower levels of their inhibitors.     

Late corneal perforation after photorefractive keratectomy associated with topical diclofenac: involvement of matrix metalloproteinases

OBJECTIVE: To report a case of a 50-year-old man who was initially seen with a corneal perforation in his right eye 2 months after a photorefractive keratectomy (PRK) procedure and to discuss the roles of topical diclofenac and matrix metalloproteinases (MMPs). DESIGN: Case report with tissue analysis. MAIN OUTCOME MEASURES: Ocular examination, diagnostic workup, surgical treatment, and histologic, immunofluorescent, zymography, and real time-polymerase chain reaction studies on corneal button. RESULTS: Slit-lamp examination of the right eye revealed a 4-mm diameter area of central corneal thinning with a 2-mm diameter perforation at its center. Predisposing factors included prolonged postoperative topical diclofenac therapy for more than 2 months and a 10-year history of well-controlled diabetes mellitus. An extensive diagnostic workup ruled out a systemic autoimmune disease. A penetrating keratoplasty was performed. Results of immunohistochemical studies of the corneal button showed stromal accumulation of temporary type III and IV collagens, MMP-3, and MMP-9 in the anterior wounded stroma and MMP-9 in the basal corneal epithelial cells of the leading edge. Differential activity and expression of MMP-2 and MMP-9 were found between the central and peripheral corneal buttons. CONCLUSIONS: Prolonged use of diclofenac and diabetes mellitus might be responsible for the corneal perforation after PRK in our patient. MMP-9 and MMP-3 might be involved in delayed wound closure and corneal melting.     

Hepatocyte growth factor stimulates proliferation and migration during wound healing of retinal pigment epithelial cells in vitro

PURPOSE: A defect in retinal pigment epithelial (RPE) cells may cause dysfunction of the neural retina, so rapid recovery of differentiated RPE cells is required after RPE injury. We investigated the effect of hepatocyte growth factor (HGF) on wound healing in RPE cells. METHODS: Confluent monolayers of bovine RPE cells were denuded, and the cells were allowed to recover in the presence or absence of HGF. The effect of HGF on RPE cell proliferation was evaluated by a 3-(4;5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetraz olium assay. In a migration assay, mitomycin C was used to inhibit proliferation, and the number of migrated cells was counted. The signaling pathways involved were examined using inhibitors of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase and protein kinase C pathways. RESULTS: At 80 ng/mL, HGF stimulated the wound closure of RPE monolayers and rendered the restituted cells more epithelioid in shape. HGF at 10 ng/mL stimulated RPE cell migration the most, whereas 80 ng/mL of HGF inhibited migration, but stimulated proliferation the most. In particular, PI3 kinase and MAPK inhibitor inhibited PRE cell migration and proliferation, respectively. CONCLUSIONS: HGF stimulated wound closure in cultured RPE cells, and rendered restituted cells epithelioid in shape. HGF may become a therapeutic candidate for RPE wound healing.     

Expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinase 1 and 2 in inflammation-induced corneal neovascularization.

PURPOSE: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMP-2, MMP-9 and TIMPs in the corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9 and TIMPs during the course of cauterization-induced corneal neovascularization in a rat model. METHODS: Neovascularization of rat corneas was induced by silver nitrate cauterization. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 was examined by immunohistochemistry and RT-PCR. The protein activities of MMPs and TIMPs were compared in pre- and postcauterization corneas by gelatin zymography and reverse zymography, respectively. RESULTS: MMP-2, TIMP-1 and TIMP-2 immunoreactivities were expressed in normal corneas, predominantly in the corneal epithelium. After injury, immunoreactivities of both MMPs and TIMPs were increased, notably in the healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts and ingrowing vascular endothelial cells. The increase in gross MMP-2 enzymatic activity paralleled the maximal vascular ingrowth on day 4, while the gross MMP-9 enzymatic activity rose immediately on day 1, then decreased steadily, which paralleled the magnitude of inflammatory cell infiltration. The immunoreactivity of MMPs/TIMPs decreased significantly 2 weeks after cauterization. On day 35, MMP-2, TIMP-1 and TIMP-2 staining was seen only in corneal epithelium and vascular endothelial cells. Both the RT-PCR and reverse zymography results revealed a more constant expression of TIMP-2, while the TIMP-1 expression appeared to be more inducible. CONCLUSION: MMPs as well as TIMPs were upregulated in cauterization-induced corneal neovascularization, suggesting that both may participate in extracellular matrix remodeling in the corneal wound healing, inflammation and neovascularization processes.     

反义寡核苷酸对人视网膜色素上皮细胞迁移的影响

目的探讨脂质体介导表皮生长因子(epidermal growthfactor receptor,EGFR)反义寡核苷酸对人视网膜色素上皮(retinal pigment epithelium,RPE)细胞迁移的影响。方法采用Murphy细胞计数方法评价反义寡核苷酸对人RPE细胞体外迁移的影响;分别应用ELISA法和半定量PCR检测反义寡核苷酸对RPE细胞EGFR蛋白和mRNA的抑制作用。结果脂质体介导EGFR反义寡核苷酸组与对照组比较,细胞迁移活性受到明显抑制(P<0.05),24h后抑制率75.27%.转染24h后EGFR蛋白和mRNA表达的抑制率分别为67.60%和35.20%.结论脂质体介导EGFR反义寡核苷酸可抑制EGFR蛋白表达和mRNA的表达,并可抑制人RPE细胞体外迁移,脂质体介导的反义寡核苷酸可能是针对RPE的一种有希望的基因治疗方法。     

Expression of metalloproteinases from human retinal pigment epithelial cells and their effects on the hydraulic conductivity of Bruch's membrane

PURPOSE: To evaluate the expression pattern of matrix metalloproteinases (MMPs) from retinal pigment epithelial (RPE) cells in culture, and to determine their ability to alter the transport properties of human Bruch's membrane. METHODS: Human RPE cells from either primary cultures or a cell line were maintained under culture conditions. At different time intervals after subculturing of cells the presence of MMPs in the bathing medium was determined by zymography. Cellular MMP activity was determined in a similar series of experiments where serum was omitted from the culture medium. Cultured cells were introduced onto Bruch's membrane, mounted in a modified Ussing chamber, to assess entry of MMPs into the membrane. Fluid flow across Bruch's membrane was determined by hydraulic conductivity for different ages of donor tissue, before and after 24 hours' incubation with active MMPs from the RPE-conditioned medium or after incubation with purified activated MMPs. Latent (inactive) MMPs from medium containing serum were used in control experiments. RESULTS: Cultured RPE cells expressed both MMP-2 and -9, with active MMP-2 becoming detectable from 4 days after subculture through to confluence and activated MMP-9 becoming abundant up to 24 hours after subculture. Both active MMPs significantly increased hydraulic conductivity of Bruch's membrane, with the increase after MMP-9 incubation being far greater than that for MMP-2. Both enzymes showed a trend in hydraulic conductivity change with age such that, MMP-2 produced a relatively constant change, whereas MMP-9 showed a greater increase in older eyes. CONCLUSIONS: Activation of both MMP-2 and -9 by cultured RPE cells appeared to show a temporal relationship with the growth cycle of the cells. The activated enzymes increased fluid flow of Bruch's membrane, and the marked effect observed with MMP-9 in older eyes suggests a mechanism that may allow debris removal.