Morphologic effects of contact lens wear on the corneal surface.

We used specular microscopy of the corneal epithelium to examine 29 eyes of 29 patients each wearing one of five different types of contact lenses. We compared these with 24 eyes of 24 age-matched control patients. We found patients with aphakic extended wear soft contact lenses had significantly larger cells (818 +/- 186 microns2) than all other groups; and they were significantly larger than their age-matched control group (573 +/- 174 microns2) (P less than .002). The epithelial cells of extended wear soft contact lens patients (609 +/- 97 microns2) and daily wear rigid gas permeable contact lens patients (613 +/- 103 microns2) were larger than their control group of normal young patients (513 +/- 53 microns2). The cells of daily wear soft contact lens patients (484 +/- 111 microns2) and hard contact lens patients (517 +/- 46 microns2), however, were not different from controls. This study demonstrates a statistically significant shift in mean cell area of corneal epithelial cells in patients wearing some types of contact lenses.     

角膜原位癌起源于角膜上皮干细胞的实验研究

目的研究角膜原位癌的组织病理学特性,探讨角膜上皮干细胞在角膜原位癌发生过程中的作用。方法收集8例角膜原位癌标本,常规石蜡包埋切片,HE染色,做形态学检查;并对其做P63和AE5免疫组化研究。结果病理检查发现角膜原位癌的癌细胞在上皮内高度增生并呈现明显的异形性,增生的细胞主要为基底细胞,上皮下基底膜完整。当用角膜上皮干细胞特异性标记物P63免疫组织化学染色,发现肿瘤区已发生癌变的恶性细胞,细胞核均染成棕色,但外围或表浅的已分化的梭形细胞未见表达,与其邻近的正常角膜上皮细胞也呈阴性反应。结论角膜原位癌细胞主要由表达P63阳性的肿瘤干细胞和少许不表达P63但表达角质蛋白的分化细胞构成,角膜原位癌起源于角膜上皮干细胞。     

Differential contributions of impaired corneal sensitivity and reduced tear secretion to corneal epithelial disorders

Background/aims  

To determine the possible roles of impaired corneal sensitivity and reduced tear secretion in various types of corneal epithelial disorders.     

Transforming growth factor-beta modulates effects of epidermal growth factor on corneal epithelial cells.

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-beta caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-beta into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-beta itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-beta itself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-beta serves as a modulator of the effects of EGF.     

Differential effects of epidermal growth factor and interleukin 6 on corneal epithelial cells and vascular endothelial cells.

PURPOSE: Epidermal growth factor (EGF) and interleukin 6 (IL-6) stimulate corneal epithelial wound healing. When applied to the cornea, these cytokines act on various types of cells and therefore may induce corneal neovascularization. We investigated the effects of EGF and IL-6 on cell proliferation and cell migration in rabbit corneal epithelial cells and human umbilical vein endothelial cells (HUVECs). METHODS: Corneal epithelial cells or HUVECs were cultured with EGF or IL-6 in the presence of 1% fetal bovine serum, and the number of cells were counted, or the radioactivity of [3H]thymidine-incorporated cells was measured. Monolayered cultured corneal epithelial cells or HUVECs were mechanically wounded, and then the cells were cultured with serum-free basal medium containing EGF or IL-6. After 12 or 24 h, the wounded area was measured. Corneal blocks were cultured with serum-free TC-199 medium containing EGF or IL-6 for 24 h, and then the length of the path of the corneal epithelium was measured. RESULTS: Estimated cell count and [3H]thymidine uptake showed that EGF stimulated cell proliferation in both corneal epithelial cells and HUVECs in a dose-dependent manner. In contrast, IL-6 did not affect cell proliferation in either cell type. Furthermore, EGF also stimulated cell migration by increasing the monolayer and organ-culture system in both cells in a dose-dependent fashion. However, IL-6 stimulated cell migration only in corneal epithelial cells and not in HUVECs. CONCLUSION: These results demonstrated that EGF stimulated cell proliferation and migration in both corneal epithelial cells and HUVECs. In contrast, IL-6 stimulated only corneal epithelial cell migration and did not affect cell proliferation in either cell type or cell migration in HUVECs. These results suggest that, when applied to the cornea, EGF might induce corneal neovascularization, and IL-6 probably would not.     

Morphologic and immunophenotypic heterogeneity of corneal dendritic cells

The morphology, distribution and immunophenotype of corneal dendritic cells was investigated in frozen sections of normal human cornea using a panel of monoclonal antibodies (mAbs). Throughout the cornea, epithelial dendritic cells were labelled with mAbs LN2 and LN3, directed to HLA-DR-associated antigens. In the peripheral corneal and limbic epithelium, dendritic cells with long cytoplasmic processes in a suprabasal position were identified as being OKT6⁺, whereas in the central corneal epithelium, dendritic cells with short processes were located basally and corresponded to the OKT6^– phenotype. Throughout the corneal stroma, LN2⁺ LN3⁺ OKT6^– spindle cells were found. This study discloses that corneal dendritic cells display considerable phenotypic and morphologic heterogeneity. We suggest that this heterogeneity is partly due to local inductive effects of peripheral corneal basal cells. Offprint requests to: B. Foets     

氧化石墨烯对大鼠角膜上皮细胞的致损伤作用

目的 探讨氧化石墨烯(oxidation of graphene,GO)对大鼠角膜上皮细胞的毒性作用.方法 体外培养Wistar雌鼠角膜上皮细胞分为正常对照组、GO干预组,正常对照组不做任何处理,GO干预组在培养时加入不同浓度(0.005 μg·L-1、0.010μg·L-1、0.020 μg·L-1、0.050 μg·L-1、0.100μg·L-1)的GO.MTT法绘制角膜上皮细胞的生长曲线、检测细胞活性;流式细胞术检测细胞的状态分布;HE染色后显微镜下观察亚细胞结构;用DAPI/PI双染以及台盼兰染色的方法进一步验证经GO干预后的细胞状态.结果 经GO干预后的角膜上皮细胞通过HE染色发现细胞内有GO的进入与残留,且细胞形态发生不规则改变,伪足部分出现纤维状结构;MTT结果显示随着GO浓度的增加,细胞活性由(81.55±1.31)％降低至(64.08±0.27)％,随着最低有效浓度的GO(O.005 μg·L-1)干预时间的增加,细胞活性由(82.61±1.36)％降低至(47.92±1.06)％;流式细胞术显示细胞破碎、死亡的比例随着GO浓度的升高,由52.9％上升至92.8％,而凋亡细胞的比例未发生明显变化;DAPI/PI双染及台盼兰染色结果均显示经GO干预后细胞死亡数目的比例急剧升高(均为P＜0.01).结论 GO对大鼠角膜上皮细胞具有毒性作用,提示GO的生物安全性存在隐患.     

Bacterial invasion of corneal epithelial cells.

PURPOSE: The normal ocular surface is frequently colonized by commensal gram-positive species. Gram-negative bacteria are often implicated in corneal infection and inflammation, particularly in association with soft contact lens wear. The aim of this study was to elucidate possible mechanisms of virulence in ocular bacteria. METHODS: The susceptibility of a human corneal epithelial cell line to bacterial invasion and association was evaluated using the gentamicin exclusion assay. Organisms tested included isolates from corneal ulcers, corneal inflammation and ocular sites in asymptomatic individuals. RESULTS: The commensal, non-pathogenic bacterium Staphylococcus epidermidis and some pathogenic strains of Serratia marcescens did not invade corneal epithelial cells. In contrast, pathogenic strains of Pseudomonas aeruginosa associated with and invaded corneal epithelial cells. CONCLUSIONS: The increased association of P. aeruginosa, compared to other bacterial types, might be a reason for the more frequent association of this bacterium with contact-lens-associated microbial keratitis.     

PM2.5对小鼠泪膜功能和角膜上皮组织结构的影响

目的 观察PM2.5对小鼠泪膜功能和角膜上皮组织结构的影响.方法 24只雄性6～8周龄BALB/c小鼠,随机分为A、B两组,每组12只.B组采用5 mg·mL-PM2.5混悬液滴眼,A组采用PBS滴眼,每天4次.分别在干预后1d、4d、7d对各组小鼠进行泪膜功能检测,包括泪液分泌功能、泪膜破裂时间(break-up time,BUT)、荧光素染色(fluorescein staining,FL),并于干预后7d行苏木精-伊红染色观察角膜上皮情况.结果 干预后4d、7d,A组泪液分泌量、BUT较干预前无明显变化,差异均无统计学意义(均为P＞0.05),而B组泪液分泌量、BUT较干预前明显分别减少和恶化,差异均有统计学意义(均为P＜0.05).干预后7d,A组FL评分较干预前无明显变化,差异无统计学意义(P＞0.05),而B组FL评分较干预前明显增加,差异有统计学意义(P=0.003).干预后7d,A组上皮细胞层数为(4±1)层,而B组上皮细胞层数为(7±1)层,差异有统计学意义(P＜0.05).与A组比较,B组整个角膜FL着染明显增加,角膜表面上皮细胞层损伤,层数增厚.结论 PM2.5会影响小鼠泪膜功能,损伤小鼠角膜上皮的组织结构.     

雄激素调节小鼠泪膜功能与角膜上皮细胞Mucins表达的体内研究

目的：探讨去势小鼠干眼模型中雄激素浓度对小鼠泪膜稳定性以及角膜上皮细胞 Mucins 表达的影响。
　　方法：通过双侧睾丸切除去势手术,改变体内雄激素水平,观察泪膜破裂时间的变化,同时在不同时间点采用RT-PCR 法与 Western-Blot 法分别检测小鼠角膜上皮细胞中 Muc1和 Muc4的 mRNA 和蛋白水平的表达变化。结果：去势1wk 后小鼠睾酮质量浓度下降至0ng/μL;正常对照组、伪手术组及去势术后1、2、4、6、8wk 组小鼠BUT 分别为68.33依12.86、62.47依3.75、58.67依10.03、47.17依7.64、39.51依3.39、32.67依3.88、31.00依2.36s,其中术后2、4、6、8wk 组小鼠泪液 BUT 值较正常对照与伪手术组均明显缩短,差异均有统计学意义(均 P<0.05)。Muc1与 Muc4的 mRNA 与蛋白水平表达,随雄激素浓度下降,表达下调(P<0.05),Muc1在去势后第2wk 表达最低,而 Muc4在去势后第1wk 表达降至最低。
　　结论：在体内,雄激素可调控小鼠角膜上皮 Mucins 表达,并缩短泪膜破裂时间,参与泪膜稳定性的维持。     

蒙脱石滴眼液对小鼠泪膜功能和角膜上皮厚度的影响

目的　研究蒙脱石滴眼液对小鼠泪膜功能和角膜上皮厚度的影响。方法　将24只BALB/C小鼠随机分成A组和B组,每组12只。A组用15 g·L^-1蒙脱石混悬液滴眼,B组用等量PBS滴眼,每天3次。分别在滴眼前和滴眼后1 d、4 d、7 d对2组小鼠进行泪液分泌试验、泪膜破裂时间（break up time,BUT）检测和角膜荧光素（fluorescein,FL）染色评估,滴眼后7 d利用光学相干断层扫描血管造影（optical coherence tomography angiograph,OCTA）进行角膜分区,分别测量2组小鼠不同区域角膜上皮厚度。结果　A组与B组泪液分泌量有差别（F=19.731,P=0.009）,A组较B组泪液分泌量低,泪液分泌破坏显著,且2组泪液分泌量的差别有随着处理时间延长逐渐变大的趋势;滴眼后4 d和7 d,两组泪液分泌量比较,差异均有统计学意义（t=3.847、5.695,P=0.026、0.009）。A组较B组BUT缩短（F=22.432,P=0.001）,泪膜破坏明显,且2组泪膜破坏程度的差别随着处理时间延长而有变大的趋势,滴眼后4 d和7 d,两组BUT比较,差异均有统计学意义（t=5.753、5.695,P=0.024、0.009）。A组较B组角膜FL染色评分增加（F=14.753,P=0.003）,角膜上皮细胞损伤明显,且2组角膜FL染色的差别有随着处理时间延长逐渐变大的趋势,滴眼后4 d和7 d,两组FL评分比较,差异均有统计学意义（t=7.536、9.864,P=0.031、0.012）。滴眼后7 d,与B组相比,A组各区域角膜上皮厚度均变厚,差异均有统计学意义（均为P＜0.05）。结论　蒙脱石滴眼液会破坏小鼠的泪液分泌功能和泪膜稳定性,并损伤小鼠角膜上皮细胞,使角膜上皮变薄。     

角膜内皮移植术后早期玻璃酸钠对泪膜及角膜上皮细胞再生的作用

目的　研究1 g·L^-1和3 g·L^-1玻璃酸钠（hyaluronate，HA）滴眼液对角膜内皮移植术后早期泪膜及角膜上皮细胞再生的作用。方法　选择2017年9月至2018年2月在北京大学第三医院行角膜内皮移植术的患者60例（60眼），随机分成1 g·L^-1HA组和3 g·L^-1HA组，每组各30例，观察标准干眼评估问卷（SPEED）评分、结膜角膜染色评分（OSS）、泪膜破裂时间（TBUT）、角膜上皮缺损范围，进行激光共聚焦显微镜检查。比较不同时间点组间、组内各指标差异。结果　SPEED评分:各时间点两组间比较差异均无统计学意义（均为P>0.05）。组内比较:两组组内术后7 d、14 d、28 d均低于前一时间点（均为P=0.000），余相邻时间点两两比较差异均无统计学意义（均为P>0.05）。TBUT:术后7 d、14 d、28 d 3 g·L^-1HA组均显著高于1 g·L^-1HA组（均为P<0.05），而术前组间差异不显著；组内比较:1 g·L^-1 HA组后一时间点较前一时间点、3 g·L^-1HA组术后7 d较术前均显著延长(均为P<0.05)，而3 g·L^-1HA组术后14 d、28 d与前一时间点差异均无统计学意义（均为P>0.05）。 OSS评分:术前及术后1 d、3 d两组间比较差异均无统计学意义（均为P>0.05），术后7 d、14 d、28 d 3 g·L^-1HA组均显著低于1 g·L^-1HA组（均为P<0.05）；组内比较:两组术后1 d OSS评分均显著高于术前（均为P=0.000），其余时间点两组均低于前一时间点（均为P=0.000）。角膜上皮缺损范围:术后1 d 两组间比较差异均无统计学意义（均为P>0.05），术后3 d、7 d 两组间比较，以及两组组内与前一时间点比较差异均有统计学意义（均为P<0.05）。角膜基底细胞密度:术前、术后28 d两组间比较，以及1 g·L^-1HA组组内术前与术后28 d比较差异均无统计学意义（均为P>0.05），而3 g·L^-1 HA组术后28 d显著低于术前（P=0.002）。结论　HA可以有效改善角膜内皮移植术后早期泪膜状态并促进角膜上皮细胞再生，存在浓度依赖性。     

Rabbit corneal epithelial cells adhere to two distinct heparin-binding synthetic peptides derived from fibronectin.

Fibronectin plays an important role in corneal reepithelialization during corneal wound healing. In this study, rabbit corneal epithelial (RCE) cell adhesion to fibronectin was further defined using proteolytic fragments of fibronectin and chemically synthesized peptides derived from the amino acid sequence of fibronectin. RCE cells adhere to intact fibronectin, the 75 kD fragment containing the RGDS (Arg-Gly-Asp-Ser) cell adhesion-promoting sequence, and the 33/66 kD cell adhesion promoting/heparin-binding fragments of fibronectin. The 75 kD fragment and the 33/66 kD fragments partially inhibited RCE cell adhesion to intact fibronectin, suggesting that these fragments represent distinct sites used by RCE cells to adhere to intact fibronectin. Two chemically synthesized peptides derived from the amino acid sequence of the 33/66 kD fragments of fibronectin, FN-C/H-I (YEKPGSPPREVVPRPRPGV) and FN-C/H-III (YRVRVTPKEKTGPMKE), directly promoted the adhesion of RCE cells. As further evidence that FN-C/H-I and FN-C/H-III play a role in the adhesion of RCE cells to the 33/66 kD fragments of fibronectin, we have shown that soluble FN-C/H-I and FN-C/H-III inhibited RCE cell adhesion on surfaces coated with the 33/66 kD fragments. In addition, polyclonal IgG against FN-C/H-I and FN-C/H-III partially blocked RCE cell adhesion to the 33/66 kD fragments, confirming that these sequences represent adhesion-promoting sites within these fragments. In contrast, two previously described peptides from the 33/66 kD fragments of fibronectin, which promoted the adhesion of a variety of cell types, FN-C/H-II (KNNQKSEPLIGRKKT) and CS-1 (DELPQLVTLPHPNLHG-PEILDVPST), did not support RCE cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration-dependent effects of lidocaine on corneal epithelial wound healing.

Local anesthetic toxicity is a recognized clinical problem that has limited the use of topical corneal anesthetics for pain relief after corneal abrasion. Studies have shown clinically administered concentrations (0.5-2%) of local anesthetics impair corneal reepithelialization. Unfortunately, instillation of local anesthetic drops into an eye does not provide a measurable, steady-state concentration of drug. Thus, it has not been possible to evaluate whether there is an analgesic concentration of local anesthetic that does not impair corneal wound healing. Using the new in vitro rabbit cornea wound healing model, the effect of steady-state lidocaine concentrations on epithelial wound healing was examined. At lidocaine concentrations below 100 micrograms/ml, wound healing was not impaired. Higher concentrations (250-1000 micrograms/ml) resulted in dose-dependent impairment of epithelial wound healing. Combined with electrophysiologic evidence that corneal nerve injury discharge can be abolished by lidocaine concentrations less than 100 micrograms/ml, this research suggests that topical lidocaine in low concentration may be a safe topical corneal analgesic.     

In vitro effects of three blood derivatives on human corneal epithelial cells

Purpose. We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. Methods. The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. Results. We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. Conclusions. PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.     

Toxicity of natural tear substitutes in a fully defined culture model of human corneal epithelial cells

PURPOSE: Serum and saliva have recently been advocated as natural tear substitutes for intractable aqueous-deficient dry eyes, but the effects of these fluids on corneal epithelium have not been well characterized. A laboratory study was performed in a defined test model to compare the toxicity of natural and pharmaceutical tear substitutes and to identify potentially toxic factors in natural tear substitutes, such as amylase, hypotonicity, and variations in preparation. METHODS: Primary human corneal epithelial cells were cultured with defined keratinocyte serum-free medium. The cells were incubated with hypromellose (hydroxypropylmethylcellulose 0.3%) with and without benzalkonium chloride 0.01%, saliva with differing osmolalities, 100% serum, and 50% serum (1:1 vol/vol with chloramphenicol 0.5%) for varying times and concentrations. Toxicity was examined in four ways. Microvillous density was assessed with scanning electron microscopy. Cell membrane permeability and intracellular esterase activity were analyzed after staining with fluorescent calcein-AM/ethidium homodimer and cellular adenosine triphosphate (ATP) was quantified using a luciferin-luciferase-based assay. RESULTS: The toxicity ranking of the tear substitutes correlated in all assays. The ATP assay was the most sensitive, followed by ethidium cell permeability, and finally the esterase activity. Preserved hypromellose was more toxic than the unpreserved preparation. Among natural tear substitutes, natural saliva was most toxic. Isotonic saliva and 50% serum were of similar toxicity, and 100% serum was least toxic. Natural tear substitutes were-except for natural saliva-less toxic than unpreserved hypromellose. Hypotonicity, but not amylase, was the major toxic effect associated with saliva. The dilution of serum with chloramphenicol induced toxicity. CONCLUSIONS: This is the first toxicity study using human primary corneal epithelial cells cultured under fully defined conditions as an in vitro model. Cellular ATP is a sensitive parameter for quantifying toxicity. Isotonic saliva and serum offer greater therapeutic potential for severely aqueous-deficient dry eyes than do pharmaceutical tear substitutes.