Effects of the COOH-terminal tripeptide α-MSH11–13 on corneal epithelial wound healing: Role of nitric oxide

It is known that α-melanocyte stimulating hormone (α-MSH) may exert anti-inflammatory effects and facilitate reparative processes in different tissues. The effective message sequence of α-MSH resides in the COOH-terminal tripeptide α-MSH_11–13. This study was undertaken to investigate the effects of topical administration of the COOH-terminal tripeptide sequence of α-MSH (α-MSH_11–13, KPV) on corneal epithelial wound healing in rabbits and the possible role of nitric oxide (NO) in these effects. The whole corneal epithelium was denuded in both eyes by mechanical abrasion. The area of the corneal epithelial defect was stained with fluorescein, photographed, and then measured before the treatment and every 12 h by a computerized software. The mean epithelial wound area and the mean percent of epithelial defect remaining at each follow-up control were compared between experimental groups. Rabbits were topically treated with KPV 1, 5 or 10 mg/ml (30 μl), two drops four times in a day, for 4 days, starting immediately after corneal abrasion, while control animals received topical phosphate-buffered saline as vehicle. In order to study the role of NO in corneal repair processes, the NO donor, sodium nitroprusside (SP, 10 mg/ml, 30 μl) was administered in both eyes, two drops four times in a day, for 4 days. The effects of KPV or SP were challenged by pre-treatment with the nitric oxide synthase inhibitor, Nω-nitro-l-arginine methyl ester (l-NAME, 10 mg/ml, 30 μl) 30 min prior to KPV or SP instillation. The mean percent epithelial defect remaining each time was significantly smaller in animals treated with KPV or SP in comparison to controls. Sixty hours later, eight out of eight (100%) corneas treated with KPV or SP were completely re-epithelized (P < 0.05) while none of the corneas treated with placebo were re-epithelized. Pre-treatment with l-NAME inhibited the facilitating effect of KPV on corneal epithelial wound healing process and totally prevented the effect of SP. Rabbit corneal epithelial cells (RCE) in culture were exposed for 1, 6 and 24 h to different KPV concentrations (0.1, 1 and 10 μM) in medium containing 15% foetal bovine serum (FBS). Cell viability was stimulated by 1 and 10 μM concentrations of the substance. Thus, KPV may facilitate corneal epithelial wound healing in rabbits with a mechanism that may involve NO disposition in corneal tissue. However, it is not known whether this mechanism is likely to depend on a direct stimulating repairing activity shared by the entire molecule of α-MSH.     

整合素在角膜上皮创伤愈合中的研究进展

整合素作为一类重要的细胞黏附分子,通过影响细胞的形态,介导细胞的黏附、迁移和增生,在角膜上皮创伤愈合中发挥了重要的作用。讨论α2β1、α3β1、α5β1、αvβ3、α6β4、α9β1和αvβ6这7种整合素在角膜上皮创伤愈合中的研究进展及其临床意义。α6β4整合素为半桥粒的主要组成部分,介导角膜上皮细胞在细胞外基质上的静态黏附,损伤后该黏附就转变为α2β1、α3β1整合素介导的动态黏附,细胞在黏附-去黏附的过程中实现迁移,从而修复创面。α6β4、α3β1整合素相互协调作用,实现上皮细胞的板层状运动。研究还发现α6β4、α3β1整合素的活化还能促进细胞的增生。损伤后上皮细胞表面α5β1、αvβ3整合素的表达上调,二者与黏着斑的形成密切相关。α9β1和αvβ6为近年来新发现的与角膜上皮创伤愈合有关的整合素,其具体作用尚有待进一步的研究。     

表皮生长因子对碱烧伤角膜伤口愈合的实验研究

目的　观察鼠表皮生长因子 (m ouse epiderm al grow th factor,m EGF)对角膜上皮创伤修复的作用 ,并探讨其量效关系。方法　采用兔角膜碱烧伤后上皮缺损模型 ,分别给予 0 .5、1、2 g· L- 1m EGF滴眼液治疗 ,生理盐水作对照组 ,每日荧光染色裂隙灯观察、记录 ,疗程 1周。结果　 2 g· L - 1m EGF治疗组与其他 3组有显著性差异 ,0 .5及 1g· L- 1m EG F与对照组之间无显著性差异。结论　尽管量效关系复杂 ,2 g· L- 1m EGF对角膜碱烧伤后上皮缺损的修复起到一定的促进作用。     

糖尿病患者角膜上皮损伤愈合延迟的研究进展

糖尿病患者角膜上皮损伤后愈合延迟较常见,持续不愈合的角膜损伤可导致复发性上皮糜烂、角膜溃疡甚至角膜穿孔,影响视功能。本文就糖尿病患者角膜上皮损伤愈合延迟的病理基础、发病机制以及相关治疗的研究进展做一综述。概述了糖尿病患者角膜上皮结构和功能的改变,阐述了高糖、蛋白酶、泪膜、生长因子和细胞因子、角膜神经以及microRNA等在发病机制中的作用,总结了治疗糖尿病患者角膜上皮损伤愈合延迟的最新思路。     

P-选择素对角膜上皮创伤修复及其中性粒细胞迁移的影响

目的观察P-选择素对角膜上皮创伤修复及其中性粒细胞迁移的影响。方法选用P-选择素基因敲除小鼠与野生型小鼠进行对比,观察角膜上皮创伤修复过程,同时用免疫组织化学方法观察中性粒细胞在角膜的迁移,并用酶联免疫吸附测定方法检测角膜中细胞因子和趋化因子的变化。结果与野生型小鼠相比,P-选择素基因敲除小鼠的角膜创伤修复延迟,向角膜迁移的中性粒细胞数量减少;中性粒细胞的迁移与趋化因子和细胞因子有密切的关系。结论P-选择素表达缺陷可导致角膜上皮创伤修复的延迟。这种延迟可能与创伤后中性粒细胞向创伤区迁移数量减少有关。     

重组人生长激素对兔角膜损伤早期修复的研究

目的：探讨重组人生长激素(rHGH)对兔角膜上皮损伤早期的修复作用。

方法：选取32只健康雄性新西兰兔制作双眼角膜上皮损伤模型,随机选取1眼滴无菌生理盐水作为对照组,另1眼滴20nmol/L重组人生长激素作为rHGH组,分别于造模前、造模后24、48、72h采用荧光素钠染色法观察角膜上皮愈合情况,采用Cochet-Bonnet角膜知觉测量仪测量检查中央角膜敏感度,同时收集各时间点泪液检测炎症因子的表达。

结果：造模后48h对照组和rHGH组角膜上皮愈合率分别为62.52%±6.73%和79.62%±10.62%(P<0.05),造模后72h分别为90.56%±9.57%和98.43%±3.65%(P<0.05)。造模后48h,对照组和rHGH组中央角膜敏感度分别为3.22±0.42、4.22±0.26cm(P<0.05)。造模后各时间点两组泪液TNF-α和IL-1α表达均较造模前增加,且对照组均高于rHGH组。造模后24、48h,两组泪液MMP-9表达均较造模前增加,且造模后48h对照组高于rHGH组(均P<0.05)。造模后24、48h两组泪液IL-21表达均较造模前增加(P<0.05)。造模后各时间点两组泪液IL-17α、Leptin表达与造模前相比及两组之间比较均无差异(P>0.05)。

结论：重组人生长激素有利于兔角膜上皮损伤早期修复。     

FK506与环孢霉素A对角膜损伤愈合的对比研究

目的　探讨他克莫司 (FK5 0 6 )对兔角膜上皮愈合的影响并与环孢霉素A(CsA)对比。方法　 18只新西兰白兔建立角膜上皮损伤模型 ,分 3组 ,每组 6只 ,随机分为 4只和 2只 ,均于去除角膜上皮后即照像 1次 ,4只兔右眼滴 1g·L-1FK5 0 6眼液 ,左眼滴 5g·L-1CsA眼液 ,另 2只兔眼不滴眼液。 3组分别于滴眼后 12、2 4、36、4 8、72h各照像 1次。结果　统计学检验显示FK5 0 6组与CsA组及空白组相比较均有显著性差异 (P <0 .0 5 ) ;而CSA组与空白组无显著性差异。结论　局部应用 1g·L-1FK5 0 6滴眼液能明显缩短兔角膜上皮愈合时间 ;而 5g·L-1CsA对角膜上皮愈合无影响。     

Genomics of corneal wound healing: a review of the literature

Corneal wound healing is a complex process: its mechanisms and the underlying genetic control are not fully understood. It involves the integrated actions of multiple growth factors, cytokines and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells and lacrimal gland cells. Following an epithelial insult, multiple cytokines are released triggering a cascade of events that leads to repair the epithelial defect and remodelling of the stroma to minimize the loss of transparency and function. In this review, we examine the literature surrounding the genomics of corneal wound healing with respect to the following topics: epithelial and stromal wound healing (including inhibition); corneal neovascularisation; the role of corneal nerves in wound healing; the endothelium; the role of aquaporins and aptamers. We also examine the effect of ectasia on corneal wound healing with regard to keratoconus and following corneal surgery. A better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design treatments that selectively modulate key phases of the healing process resulting in scars that more closely resemble normal corneal architecture.     

P38 MAPK在角膜创伤愈合过程中的作用

丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)级联是细胞内重要的信号传导系统。细胞通过这一信号传导系统将细胞外信号传递到细胞核内,介导细胞产生反应,调节细胞生长、增殖、分化和凋亡等生理过程及创伤愈合和炎症反应等病理过程。近年来发现一类新的MAPK通路—p38MAPK信号通路,对其结构和功能以及在角膜创伤愈合修复和炎症反应中的作用已有了进一步的了解,在信号通路水平调控p38MAPK的表达和活性,可能成为临床角膜创伤治疗的新途径。     

Comparison of cytotoxicity and wound healing effect of carboxymethylcellulose and hyaluronic acid on human corneal epithelial cells

Abstract AIM: To investigate the cytotoxic effect on human corneal epithelial cells (HCECs) and the ability to faciliate corneal epithelial wound healing of carboxymethylcellulose (CMC) and hyaluronic acid (HA). METHODS: HCECs were exposed to 0.5% CMC (Refresh plus®, Allergan, Irvine, California, USA) and 0.1% and 0.3% HA (Kynex®, Alcon, Seoul, Korea, and Hyalein mini®, Santen, Osaka, Japan) for the period of 30min, and 4, 12, and 24h. Methyl thiazolyl tetrazolium (MTT)-based calorimetric assay was performed to assess the metabolic activity of cellular proliferation and lactate dehydrogenase (LDH) leakage assay to assess the cytotoxicity. Apoptotic response was evaluated with flow cytometric analysis and fluorescence staining with Annexin V and propiodium iodide. Cellular morphology was evaluated by inverted phase-contrast light microscopy and electron microscopy. The wound widths were measured 24h after confluent HCECs were scratch wounded. RESULTS: The inhibitory effect of human corneal epithelial proliferation and cytotoxicity showed the time-dependent response but no significant effect. Apoptosis developed in flow cytometry and apoptotic cells were demonstrated in fluorescent micrograph. The damaged HCECs were detached from the bottom of the dish and showed the well-developed vacuole formations. Both CMC and HA stimulated reepithehlialization of HCECs scratched, which were more observed in CMC. CONCLUSION: CMC and HA, used in artificial tear formulation, could be utilized without any significant toxic effect on HCECs. Both significantly stimulated HCEC reepithelialization of corneal wounds.     

血小板源性生长因子与角膜损伤修复

血小板源性生长因子(PDGF)在机体伤口愈合过程中具有重要的调节作用。由于角膜的特殊结构和功能,其伤口愈合的调节显得至关重要。本文简述PDGF的结构、受体、生物学活性及其在角膜伤口愈合中的作用。     

肠道真菌菌群失调对小鼠角膜创伤修复的影响

目的：观察抗真菌药物诱导的肠道真菌菌群失调对小鼠角膜创伤修复的影响。

方法：选用健康无眼疾的C57BL/6J雄性小鼠,将实验小鼠随机分为两组：对照组和两性霉素B组。对照组给予正常饮食,两性霉素B组给予添加两性霉素B的饮食,以诱导小鼠肠道真菌菌群失调,4wk后对两组小鼠角膜进行上皮创伤,使用荧光素钠染色角膜创伤区域,动态观察上皮修复情况,并利用免疫荧光染色观察角膜上皮细胞、炎症细胞变化,通过HE染色观察角膜厚度的变化。

结果：两性霉素B组与对照组相比,再上皮化速度和创伤修复延迟,炎症细胞明显减少,角膜上皮厚度变薄。

结论：肠道真菌菌群失调延迟角膜创伤的修复,降低角膜创伤后的炎症反应。     

In vivo fluorescent labeling of corneal wound healing fibroblasts

Numerous studies have shown that fibroblasts play an important role in corneal wound healing, however, the dynamic cellular events underlying wound tissue organization and contraction remain unclear. The purpose of this study was to develop a system to enable live cell imaging of corneal wound healing fibroblasts in situ. To this end, concentrated preparations of an RD114 pseudotyped MLV-based vector expressing the enhanced green fluorescent protein (EGFP) were evaluated in vitro for gene transfer efficiency using cultured rabbit corneal keratocytes. Primary rabbit keratocytes were efficiently labeled in vitro (up to 50% EGFP(+)) at a low multiplicity of infection (MOI=10). To evaluate this gene transfer vector in vivo, rabbit corneal fibroblasts were transduced by direct application of vector supernatant to injured corneas following lamellar keratectomy. Fluorescent fibroblasts were then visualized in situ using epifluorescence microscopy and multiphoton confocal microscopy of excised fresh tissue at multiple time points from 14 days to four months following gene transfer. Fourteen days post-transduction, labeled fibroblasts expressing EGFP were readily detectable by fluorescence microscopy. Detectable fluorescence was noted up to eight weeks post-transduction. Labeled fibroblasts were detected in clusters located predominantly along the margin circumscribing the wound and to a lesser extent within the wound area. Cell growth in clusters was suggestive of the expansion of individual transduced clones. High-resolution imaging showed fluorescent fibroblasts to have a broad, flattened, dendritic morphology, distinct from the spindle shape of cultured fibroblasts. Utilizing multiphoton confocal microscopy, three-dimensional imaging of viable, labeled cells showed wound healing fibroblasts to be extensively interconnected and multi-layered within the corneal wound. These results demonstrate that rabbit corneal fibroblasts can be efficiently transduced in vitro and in vivo using RD114 pseudotyped MLV-based vectors and that these vectors direct long-term transgene expression without apparent toxicity, pathogenesis or perturbation of native fibroblast morphology. Our data further suggest that, in vivo, wound-healing fibroblasts have a defined life span within the wound.     

Anti-angiogenic role of angiostatin during corneal wound healing

The purpose of this study is to determine whether angiostatin is involved in maintaining corneal avascularity after wounding. We generated polyclonal rabbit anti-mouse angiostatin antibodies directed against each of the five kringle domains, (K1-5) and anti-mouse plasmin B chain antibodies. Mouse corneas were immunostained with anti-K1 angiostatin antibody after excimer laser keratectomy. Corneal epithelial cell lysate was harvested and angiostatin was isolated using lysine sepharose. Purified plasminogen was incubated with lysate of mouse corneal epithelial cells from wild type mice in the presence or absence of MMP inhibitors. Angiostatin activity was determined using calf pulmonary artery endothelial (CPAE) cell proliferation assay with and without angiostatin immunoprecipitation; and corneal neovascularization was assayed by intrastromal injection of anti-plasminogen, anti-K1-3 or anti-B chain antibodies after corneal wounding. Using the anti-mouse angiostatin antibodies that we generated, we confirmed that angiostatin-like molecules were expressed in the corneal epithelium and in cultured corneal epithelial cells. Western blotting after incubation of scraped corneal epithelial cell lysate with purified plasminogen showed reduction of the plasminogen bands at 6, 12, and 24 hr, respectively. Complete cleavage of plasminogen occurred by 48 hr. Functional assays in which corneal epithelial cell extracts were incubated with CPAE cells resulted in inhibition of vascular endothelial cell proliferation. Depletion experiments using anti-angiostatin (K1) antibodies resulted in a 25 +/- 1.2% increase in vascular endothelial cell proliferation as compared to 12 +/- 1.8% using the protein A control (p < 0.05). Corneal neovascularization was observed after excimer laser keratectomy when anti-angiostatin antibodies were injected into the cornea (65 +/- 13%) which was significantly higher than when plasmin B chain antibodies were injected (10 +/- 2.6%; p < 0.05). Plasminogen and angiostatin are produced in the cornea. They may play a role in preventing vascularization and may contribute to the maintenance of corneal avascularity after excimer laser keratectomy.     

血小板源生长因子与角膜的损伤及修复

角膜损伤是眼科常见病,常致视力严重损害。角膜损伤的修复是一个复杂的过程,多种细胞因子参与其中。血小板源生长因子可能在角膜损伤的修复过程中起重要作用。研究血小板源生长因子与角膜的关系,特别是其在角膜损伤后修复过程中的作用及机制,可能会对角膜疾病的防治开辟一条崭新的有效途径。     

Influence of cell and collagen concentration on the cell-matrix mechanical relationship in a corneal stroma wound healing model

The effect of different collagen and cell concentrations on the mechanical and remodeling behaviors of corneal stroma wound healing models consisting of collagen hydrogels seeded with human corneal fibroblasts during a 25 day culture period were examined. Human corneal fibroblasts were seeded at 1 × 10⁵, 3 × 10⁵ or 5 × 10⁵ cells per hydrogel, and collagen concentrations of 2.5 mg/ml, 3.5 mg/ml or 4.5 mg/ml were examined. Two non-destructive techniques, spherical indentation and optical coherence tomography, were used to measure the elastic modulus and dimensional changes respectively at several time-points over the culture period. The elastic modulus of the hydrogels increased continuously over 25 days. Hydrogels with higher initial cell seeding densities and lower initial collagen concentrations were found to increase in elastic modulus faster and possessed a higher elastic modulus by the end of the culture period when compared to the other hydrogels. A mathematical equation was applied to accurately fit the change in elastic modulus over time. This study demonstrates a robust in vitro technique able to monitor the effect of different parameters on the cell-matrix mechanical relationship in a corneal stroma model during prolonged culture periods and enhances our understanding on corneal wound healing processes.     

高糖对人角膜上皮细胞创伤修复中Cyclin D1 蛋白表达的影响

目的： 研究高糖环境对人角膜上皮细胞损伤修复的影响,初步探讨Cyclin D1蛋白在高糖培养的角膜上皮细胞创伤愈合中表达的意义。

方法： 模拟糖尿病角膜病变的高糖微环境,人角膜上皮细胞经复苏、培养传代后,分别设置等体积蒸馏水的DMEM完全培养基的正常对照组和含25mmol/L葡萄糖的DMEM完全培养基高糖处理组的两组细胞,细胞长满后进行划伤刺激,倒置相差显微镜下观察比较各组细胞生长状况及其变化,于不同时间点(0、12、24、48和72h)利用Western blot检测分析高糖培养的角膜上皮细胞中Cyclin D1蛋白的表达,qRT-PCR检测Cyclin D1 mRNA水平相对表达情况。

结果： 体外高糖处理条件下,人角膜上皮细胞划伤后修复速度减慢,漂浮细胞增多,细胞重新贴壁少,细胞间距增加,随着高糖处理时间的延长,细胞状态变差,生长速度慢; 正常组细胞损伤修复较快, 细胞密度增大,且形态规则,细胞膜光滑。Western blot 检测划伤刺激上调Cyclin D1的表达,但随着时间延长上调作用呈逐渐减弱,两组Cyclin D1最高表达量均出现在12h。高糖处理组Cyclin D1表达量低于正常对照组。qRT-PCR结果显示：高糖处理后,Cyclin D1 mRNA表达呈上调趋势,但随着高糖处理时间延长,上调作用减弱,且在48h处mRNA水平恢复至与对照组同一水平。

结论： 在角膜上皮细胞创伤愈合过程中,高糖呈负性调节,抑制Cyclin D1蛋白的表达,且与角膜上皮细胞增殖能力下降及凋亡相关。     

Cellular and molecular events in corneal wound healing: significance of lipid signalling

Alterations in the normal healing process after corneal injury can produce undesirable outcomes that range from corneal haze to ulceration and perforation. Lipids play important roles in the complex inflammatory processes that occur after corneal wounding. While some lipid mediators, such as the lipoxygenase derivatives of arachidonic acid, 12-hydroxyeicosatetraenoic acid (12[S]-HETE and 15[S]-HETE), act as second messengers to promote cell proliferation and are possibly involved in the synthesis of other molecules that suppress inflammation, others, such as platelet-activating factor (PAF), exert their actions through specific receptors, play key roles during sustained corneal inflammation (as might occur with chemical burns), and contribute to tissue destruction and neovascularization. PAF is also a strong inducer of selective metalloproteinases (MMPs) that degrade the extracellular matrix. The use of a new PAF antagonist has shown great promise for the treatment of diffuse lamellar keratitis (DLK) and alkali-burned corneas.