Beta-adrenergic and serotonergic responsiveness of rabbit corneal epithelial cells in culture

Rabbit corneal epithelial cell cultures were established from Dispase-treated anterior corneas. In culture medium containing cholera toxin, insulin and epidermal growth factor, these cells proliferated in vitro in the absence of any contaminating cells. Following subculture, cells retained epithelial morphology and the ability to synthesize cAMP in response to beta-adrenergic stimulation, but lacked the ability to respond to serotonergic stimulation. Retention of the beta-adrenergic system in culture serves as a functional epithelial cell marker; whereas expression of serotonergic responsiveness may be regulated by developmental or extrapithelial systems that are absent in these cell cultures.     

Polyamines in cultured rabbit corneal cells

PURPOSE: To determine whether polyamines are present in corneal cells, whether corneal cell polyamines can be depleted by blocking the first rate-limiting enzyme in the polyamine synthesis pathway, ornithine decarboxylase (ODC), and whether polyamines are required for proliferation in all three corneal cell types. METHODS: Cultured corneal epithelial cells, keratocytes, and endothelial cells were exposed to the specific ODC blocker difluoromethylornithine (DFMO), and ODC activity, intracellular polyamine concentrations, and cell proliferation were measured. RESULTS: DFMO blocked ODC activity in a dose- and time-dependent manner in all three cell types. DFMO treatment completely depleted putrescine and spermidine by 2 days and also significantly depleted spermine. DFMO treatment also inhibited cell growth in all three cell types and this inhibition could be completely reversed by adding exogenous putrescine to the culture medium. CONCLUSIONS: Polyamines are present in all cell types of the cornea, their formation is catalyzed at least in part by ODC, and they are an important component of corneal cell proliferation.     

体外培养的兔角膜内皮细胞生物学特性

背景如何选择高质量、生物学特性更接近在体生理状态的角膜内皮种子细胞是组织工程角膜研究的基础。角膜内皮细胞（CECs）的培养、鉴定及生物学特性检测是优选角膜内皮种子细胞的瓶颈问题。目的建立兔CECs原代培养及鉴定的方法,检测传代后细胞的生物学特性。方法从30只新西兰大白兔的角膜组织完整撕除后弹力层及CECs层,采用胰蛋白酶消化法进行原代培养,观察培养细胞的生长状态。分别从形态学、基因水平和蛋白水平鉴定细胞：茜素红染色法观察细胞形态,并与新鲜角膜组织的内皮细胞进行对比;逆转录聚合酶链反应（RT—PCR）法检测IVα2型胶原（COL4A2）、血管内皮生长因子受体2（FLK1）、Na^-K^＋ATP酶α亚单位（ATPIA1）、水通道蛋白1（AQP1）、电压依丛性阴离子通道（VDACs）等相对特异性基因;免疫细胞化学法观察神经元特异性烯醇化酶（NSE）、Na^-K^＋ATP酶及紧密连接蛋白ZO-1的表达。采用MTT比色法测定传代细胞的增生活性变化,采用超微量ATP酶试剂盒及免疫荧光法分别测定传代细胞Na^-K^＋ATP酶活性及其表达量的变化。结果原代培养的兔CECs多在24h内贴壁,2—3d达融合,形态呈六边形。随着传代代数的增加,细胞形态逐渐发生改变,第2代、第3代细胞可见空泡样变。原代培养的细胞茜素红染色可见清晰的细胞轮廓,与在体的CECs形态相似。RT—PCR法检测可见COL4A2、FLKl、ATP1Al、AQP1、VDACs等基因在培养细胞中表达。免疫荧光染色结果显示NSE、Na^-K^＋ATP酶、ZO-1在培养细胞中呈阳性表达。MTT检测结果显示,传代后细胞的增生活性逐渐下降,尤其第2代、第3代细胞下降明显。定量检测结果显示随着传代代数的增加,兔CECs的Na^-K^＋ATP酶活性逐渐降低,各代细胞的Na^-K^＋ATP酶活性总体比较的差异有统计学意义（F=77．174,P=0．000）。结论成功用酶消化法建立了体外培养兔CECs的方法,并从多个层次建立了纯化细胞的鉴定方法。研究结果证实经传代后的CECs的增生活性和功能均有所下降,因此在进行相关的研究时应选用前2代的细胞。     

Chemotactic and haptotactic activities of fibronectin for cultured rabbit corneal epithelial cells

A previous study reported that both fibronectin and epidermal growth factor (EGF) stimulated corneal epithelial resurfacing. Fibronectin appears in the cornea after injury, and corneal epithelial cells migrate over the temporary fibronectin matrix. To determine whether fibronectin serves as chemoattractant and haptoattractant for the directed movement of corneal epithelial cells, the directed migration of cultured rabbit corneal epithelial cells was measured in vitro using a Boyden chamber. Chemotactic and haptotactic migration were assayed separately. Fibronectin was found to stimulate attachment of corneal epithelial cells and to have chemotactic, haptotactic and chemokinetic activities for the corneal epithelial cells. In contrast, EGF had no chemotactic activity.     

丝裂霉素对体外培养兔角膜内皮细胞的毒性影响

目的探讨丝裂霉素C(mitomycinC,MMC)对角膜内皮细胞的毒性作用。方法体外培养家兔角膜内皮细胞,接种于96孔板,加入不同浓度的MMC,分别为1×10-6g·L-1、1×10-5g·L-1、1×10-4g·L-1、1×10-3g·L-1、1×10-2g·L-1、1×10-1g·L-1,动态观察细胞的形态变化和增殖情况,加药22h后MTT法检测吸光度值,应用t检验比较各组间的差异。结果10-2g·L-1、10-1g·L-1浓度组8h后细胞部分死亡,20h细胞几乎全部死亡,其余各组变化不明显。统计学结果显示:10-6g·L-1、10-5g·L-1组和对照组比较无显著性差异(P>0.05),10-4g·L-1组有显著性差异(0.01相似文献   

Effects of isopropyl unoprostone ophthalmic solution on cultured rabbit corneal epithelial cells

PURPOSE: To investigate the effects of isopropyl unoprostone (referred to as unoprostone) ophthalmic solution on the barrier function of cultured rabbit corneal epithelium grown on permeable supports. METHODS: Rabbit corneal epithelial cells cultured on collagen-coated filter inserts were administered one of the following for 30 min: unoprostone in vehicle solution (polysorbate 80), unoprostone in vehicle solution with a preservative (benzalkonium chloride), preservative only or vehicle only. For a control, no chemicals were added to the medium. After administration, the transepithelial electrical resistance (TER) measurement, a sensitive method by which to investigate the barrier function, and morphological observation using phase-contrast microscopy were performed before exposure and at 0.5, 1, 3, 6, 12, 24, 48 and 72 h after exposure. The transmission electron-microscopic observation was performed before and 72 h after exposure in all experimental conditions. RESULTS: The cells exposed to unoprostone with the preservative showed a significant decrease in the TER, although no morphological changes were observed. The corneal epithelial cells exposed to unoprostone without preservative, the vehicle only or the preservative only did not show any differences from the control group at any measurements. CONCLUSION: The corneal barrier function is damaged by a combined solution of unoprostone and preservative, but not by a single solution of unoprostone, in vitro.     

人表皮生长因子对体外培养兔角膜内皮细胞影响的实验研究

目的：明确人表皮生长因子（ｈｕｍａｎ Ｅｐｉｄｅｒｍａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ,ｈＥＧＦ）对角膜内皮细胞的影响。方法：采用体外培养的兔角膜内皮细胞,观察接种后不同时点ｈＥＧＦ对其生长状态的影响;兔角膜片内皮损伤模型,离体培养后行Ｈ－Ｅ染色和Ｈ＾３－ＴｄＲ掺入放射自显影,观察ｈＥＧＦ对其修复的影响。结果：兔角膜内皮细胞体外培养ｈＥＧＦ组细胞数高于对照组,并使细胞形态产生纺锤形改变;角膜内皮细胞     

The uptake of fluoroquinolone agent drugs by cultured human conjunctival epithelial cells and cultured SIRC rabbit corneal cells]

PURPOSE: To investigate the uptake of drugs by cultured human conjunctival epithelial cells (HCEC) and cultured rabbit corneal cell lines (SIRC). METHOD: The drugs examined were ofloxacin (OFLX), lomefloxacin (LFLX), and norfloxacin (NFLX). The amount of drug uptake in the cultured cells that were exposed to the drugs was measured by high-performance liquid chromatography (HPLC). RESULT: The amount of LFLX, OFLX, and NFLX uptake in HCEC was 1.38 +/- 0.21 nmol/10(6) (mean +/- standard deviation) cells (5 min), 0.79 +/- 0.05 nmol/10(6) cells (5 min), and 0.56 +/- 0.03 nmol/10(6) cells (5 min), respectively. The amount of LFLX, OFLX, and NFLX uptake in SIRC was 1.08 +/- 0.18 nmol/10(6) cells (5 min), 0.58 +/- 0.17 nmol/10(6) cells (5 min), and 0.41 +/- 0.35 nmol/10(6) cells (5 min), respectively. The uptake levels of LFLX by both cultured cells were higher than those of OFLX and NFLX. CONCLUSION: This method may be useful as a screening method for intraocular drug dynamics studies.     

Intracellular potentials of cultured rabbit corneal endothelial cells: Response to temperature and ouabain

The intracellular potentials of rabbit corneal endothelial cells grown in culture were measured with KCl-filled glass microelectrodes. In a physiological salt solution at 35°C the potential difference (p.d.) across the endothelial cell membrane was ?59.2 ± 1.8mV. This p.d. was reduced to ?40.5 ± 1.3mV at 23°C. The addition of 10^?6 M ouabain to confluent cell cultures caused the p.d. to be reduced to about ? 49 mV.     

Epidermal growth factor stimulates phospholipase cgamma1 in cultured rabbit corneal epithelial cells

Phospholipase Cgamma1 (PLCgamma1) catalyses hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)), two second messengers which play important roles in cell proliferation and differentiation. The purpose of the current study was to identify PLCgamma1 in corneal epithelial cells and investigate whether epidermal growth factor (EGF) stimulates the activity of this enzyme. Addition of EGF to [(3)H]myo-inositol-labeled, cultured corneal epithelial cells stimulated production of IP(3), indicating activation of PLC. Western immunoblot analysis and an in vitro assay of PLC activity revealed that EGF activates gamma1 isoform of PLC, which is localized predominantly in the cytosolic fraction of the epithelial cells. EGF receptors were detected in the epithelial cells by EGF receptor antibody. Addition of EGF to the cells caused tyrosine phosphorylation of the receptors, translocation of PLCgamma1 from cytosol to plasma membrane, and phosphorylation of the enzyme at tyrosine residues. Addition of tyrphostin A-25, an inhibitor of receptor tyrosine kinase, attenuated the tyrosine phosphorylation of PLCgamma1 as well as its enzyme activity. These findings suggest that EGF stimulates PLCgamma1 in rabbit corneal epithelial cells, and that this effect is probably mediated by tyrosine phosphorylation of the enzyme.     

Effects of tranilast on cultured rabbit corneal keratocytes and corneal haze after photorefractive keratectomy

PURPOSE: In vitro and in vivo studies were performed to elucidate the effects of tranilast on cellular proliferation and collagen synthesis. METHODS: Subculturing was carried out using keratocytes from rabbits that underwent photorefractive keratectomy (PRK) and developed corneal haze, and keratocytes from normal rabbit cornea. RESULTS: Tranilast suppressed proliferation in cultured keratocytes from the corneal haze region at doses of 30 and 300 micromol/L and collagen synthesis at doses of 3, 30, and 300 micromol/L. Normal corneal cultures showed suppression of keratocyte proliferation and collagen synthesis only at a high dose of tranilast (300 micromol/L). Betamethasone suppressed proliferation of keratocytes in both haze and normal cornea at a dose of 10 micromol/L, as well as collagen synthesis at respective doses of 1 and 10 micromol/L. Diclofenac sodium suppressed collagen synthesis of keratocytes in haze cornea at a high dose of 100 micromol/L, and in keratocytes in normal cornea, at doses of 10 and 100 micromol/L. In an in vivo study, either 0.5% tranilast, 0.1% betamethasone phosphate eye drops, or a tranilast base solution (control) was instilled four times daily to rabbits that had undergone PRK. Weekly evaluation of the inhibitory effect of these drugs on the development of haze was performed 2 weeks after surgery. Tranilast suppressed haze 6-13 weeks after PRK, but betamethasone phosphate showed no effect. CONCLUSION: These results indicate that tranilast is potentially effective for inhibiting the corneal haze that occurs after PRK.     

低强度氦-氖激光对体外培养的兔角膜内皮细胞增殖的影响

目的探讨不同条件低强度氯氖激光照射对体外培养的兔角膜内皮细胞增生的影响。方法体外培养的兔角膜内皮细胞经低强度激光照射后，MTT比色法检测其增生活性；通过生物显微镜、组织化学染色观察其照射前后形态学变化；通过流式细胞仪检测其照射前后细胞周期的变化等。结果MTT检测结果显示，体外培养的兔角膜内皮细胞经低强度激光照射后，其增生活性提高，本实验中最适照射强度和时间分别是5mW·cm^-2、600s；流式细胞仪细胞周期检测结果显示：激光照射组的细胞增生指数明显高于对照组，而与碱性成纤维细胞生长因子组比较差别无显著性意义。结论低强度激光在一定程度上可促进角膜内皮细胞的增生。     

Adsorptive and fluid phase endocytosis by cultured rabbit corneal endothelium

In addition to maintaining corneal transparency via a pump-leak mechanism, the corneal endothelium plays an active role in the transport of certain proteins to supply nutrients for the stroma and to remove metabolites. In order to investigate transcellular transport mechanisms across the endothelium, we exposed cultured rabbit corneal endothelial cells to tracers that are commonly used to describe various forms of endocytosis. The cells were incubated on their apical surfaces in solutions containing different HRP conjugated lectins (concanavalin A and lens culinaris = alpha-D-mannose, alpha-D-glucose; peanut agglutinin = alpha-D-galactose), cationized ferritin (CF), native ferritin (NF), and HRP alone for 5-60 min and processed for EM cytochemistry. At early times, the lectins and CF were seen bound to the apical plasma membrane, thus indicating adsorptive endocytosis. The NF and the lectins in the presence of their competing sugar as well as HRP showed little or no surface binding, thus being markers for fluid phase endocytosis. At later times, large amounts of lectins and CF were located in round, tubular, or U-shaped vesicles of various sizes, large vacuoles, multivesicular bodies, and in other cytoplasmic compartments. Very little or no uptake was observed with NF, or when the lectins were used in presence of their competing sugars. HRP was seen in moderate amounts only in round or oval shaped vesicles. This study suggests that adsorptive endocytic pathways play a major role in transcellular transport through the corneal endothelium, whereas the transport of macro-molecules via fluid phase endocytosis is more limited. In addition, our observations suggest that adsorptive endocytic tracers undergo various intracellular fates and also appear to be transported through the cells at different rates.     

Glucose consumption in cultured corneal cells

The rate of glucose consumption in cultured epithelium, endothelium, and keratocytes was measured; and the effect of reduced glucose availability on the consumption rate of these three cell lines was delineated. All three cell types exhibited an asymptotic decrease of glucose over time while being incubated in Krebs-Ringers solutions of varying glucose concentrations. At a concentration resembling that of the aqueous, epithelium (EPI), endothelium (ENDO), and keratocytes (K) consumed 6.7, 7.4, and 9.0 micrograms/cm2/hr respectively. Each cell type consumed glucose at a rate that was related to the amount of available glucose. As glucose concentration was reduced from 90 to 30 mg%, which was a 66% reduction in available glucose, the consumption of EPI, ENDO, and K dropped 74%, 61%, and 44% respectively.     

Pharmacological regulation of morphology and mitosis in cultured rabbit corneal endothelium

Cultured rabbit corneal endothelial cells elongate when grown in the presence of epidermal growth factor (EGF) and indomethacin (INDO); whereas maintenance of the differentiated polygonal cell shape is apparently dependent upon endogenous synthesis of prostaglandin E2 (PGE2). In the current study, the authors demonstrate morphological changes in phenotypically altered cells and identify two intracellular pathways which interdependently regulate endothelial cells. Morphometric and mitotic analyses of cultures treated with a variety of pharmacological agents indicate that both protein kinases A- and C-dependent pathways regulate cell shape and cell division in corneal endothelial cells. Marked intracellular reorganization is associated with the morphological changes in the endothelial cells. When stained with rhodamine conjugated phallicidin, polygonal endothelial cells have circumferential bands of f-actin at their borders. EGF and/or INDO induce elongation and redistribution of f-actin into a diffuse cytoplasmic reticulum. Transmission electron microscopy demonstrates loss of several characteristic morphological markers for endothelial cells in response to pharmacologically induced elongation. The elongated cells lose intracellular junctions, apical/basal polarity and rough endoplasmic reticulum. These ultrastructural markers and circumferential f-actin bands are restored in cultures supplemented with exogenous PGE2. Modulation of these pathways in vivo may regulate cellular migration and mitosis during wound closure, stress, trauma and with age.     

刀豆蛋白A条件化培养基对体外培养兔角膜内皮细胞Na^＋-K^＋-ATP酶的作用

目的 研究刀豆蛋白A(ConA)条件化培养基对体外培养兔角膜内皮细胞膜表面Na+-K+-ATP酶的分布及其酶活性的影响.方法 超微量ATP酶试剂盒测定原代培养兔角膜内皮细胞细胞膜表面Na+-K+-ATP酶的活性,并对ConA条件化培养基作用下不同生长周期(5、7、10 d)细胞Na+-K+-ATP酶的活性进行测定;免疫酶组织化学电镜下观察不同培养基作用下体外培养角膜内皮细胞膜表面Na+-K+-ATP酶的分布.结果 ConA条件化培养基组兔角膜内皮细胞Na+-K+-ATP酶活性明显高于无ConA组(P＜0.01),原代细胞生长7～10 d时Na+-K+-ATP酶活性最高(P＜0.01).单层兔角膜内皮细胞膜外侧可见Na+-K+-ATP酶阳性反应.在ConA条件化培养基处理组,其阳性反应物较多、致密;而在无ConA组则阳性反应物较少、疏松.结论 体外原代培养角膜内皮细胞在7～10 d时,Na+-K+-ATP酶活性最高.ConA条件化培养基可提高兔角膜内皮细胞Na+-K+-ATP酶活性.     

Toxic effects of ophthalmic preservatives on cultured rabbit corneal epithelium

We investigated the effects of the ophthalmic preservatives thimerosal and sorbic acid on the proliferation and survival of rabbit corneal epithelial cells in tissue culture. Normally, explants of corneal epithelium grow vigorously during the first 7 days in culture. With 0.004% thimerosal present in the culture medium, the normal proliferation of corneal cells is suppressed completely. When 0.1% sorbic acid is present, proliferation is delayed and the lifespan of the corneal cells is reduced. After a 1-h exposure to concentrations of thimerosal of 0.0005% or greater, virtually all corneal cells present in established cultures are killed. These results suggest that use of ophthalmic preparations containing these chemicals may affect the metabolic and proliferative capacity of the corneal epithelium adversely.