人角膜基质细胞的表型研究进展

人角膜基质细胞(HCSCs)是一类呈静息状态的神经嵴间充质细胞, 是负责分泌基质的高度特化的透明组织, 在维持角膜透明度和人眼正常视觉功能等方面发挥重要作用。正常情况下, 角膜基质细胞呈静息状态并表现为扁平、树突状, 在角膜受到创伤刺激后被激活, 激活状态的人角膜基质细胞(HCK)向修复表型转变, 发生凋亡或向角膜成纤维细胞表型和肌成纤维细胞表型转化。HCSCs的表型转化与人角膜损伤修复过程所引起的瘢痕组织形成以及角膜透明度降低等方面具有密切关系。本文通过对HCSCs的3种表型、表型标志物、表型间转化以及体外转化的机制进行综述, 发现通过干预HCK向纤维化表型转化过程及TGF-β/Smad等相关信号通路可以抑制角膜纤维化。因此, 深入研究HCSCs表型转化的分子机制及干预角膜瘢痕形成的调控机制, 有助于临床防治患者角膜纤维化和角膜术后混浊。     

细胞外基质对角膜创伤修复的作用

细胞外基质在角膜创伤修复过程中起至关重要的作用，其合成代谢受生长因子的调控，两者之间的的关系是是近年来令人感兴趣的新的研究领域，它将有助于阐明角膜创伤修复机制本文概述了细胞外基质的成分、结构、功能和在角膜创伤修复中的作用以及生长因子在务修复过程中对细胞外基质的调控。随着人们对角膜基础研究认识的不断深入，证明角细胞外基质（ＥＣＭ）并非一种被动无活性的结构支架，而是一种活泼的动态物质，它影响着细胞的分     

角膜上皮和基质对角膜上皮基底膜再生的影响

基底膜是一种高度特化的细胞外基质,广泛存在于人体的各组织中。自19世纪基底膜被发现以来,人们逐渐认识了其结构和功能。角膜上皮基底膜(EBM)通过限制活化的纤维化因子,参与调控角膜瘢痕形成。角膜损伤后,角膜瘢痕的形成和持续时间由角膜EBM的损伤程度及再生速度决定。角膜上皮及基质共同参与了角膜EBM的再生。角膜上皮的迅速再...     

TGF-β在角膜瘢痕形成及无瘢痕愈合中的作用

转化生长因子β（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒ-β,ＴＧＦ-β）是一种多功能的细胞因子。体外和多种动物模型中研究已证实在角膜ＴＧＦ-β是主要参与角膜损伤后启动瘢痕化的重要生长因子。近年来针对ＴＧＦ-β抗瘢痕化研究已取得一些进展。本文综述了ＴＧＦ-β分子结构、生物学功能,角膜损伤修复中瘢痕形成过程中基质细胞在ＴＧＦ-β调控下的变化及ＴＧＦ-β治疗角膜瘢痕的进展。     

角膜基质细胞的表型转化及其预防瘢痕形成的研究

正常情况下角膜基质细胞处于静止状态，受损伤时易发生表型及生理功能的改变，转换为成纤维细胞或肌成纤维细胞。不同的损伤因子影响角膜细胞的应答反应，决定角膜组织是完全修复还是形成瘢痕组织。角膜基质细胞合成分泌多种细胞外基质，活化的角膜基质细胞利于角膜损伤修复，角膜肌成纤维细胞可致角膜表面混浊，损伤、细菌或病毒感染等因素导致角膜受损后修复产生瘢痕，富血小板血浆、转化生长因子β等生化因子在表型转换、瘢痕组织的临床治疗和预防等方面起重要作用。（国际眼科纵览， 2018,  42:   194-198）     

重视角膜基质细胞的作用

位于角膜基质胶原纤维板层之间的角膜基质细胞处于静息状态,可以分泌胶原以及硫酸角质素,对角膜透明性的维持发挥着重要作用。当角膜受到损伤时,角膜基质细胞可发生凋亡,向成纤维细胞以及肌成纤维细胞等修复细胞表型转化,进而促进细胞再生,诱导角膜纤维瘢痕形成。此外,大量角膜基质细胞还具有干细胞特性。目前已知角膜基质细胞是多种机体功能的积极参与者,应重视角膜基质细胞的作用。     

小分子亮氨酸重复蛋白聚糖家族在维持角膜透明性中的作用

小分子亮氨酸重复蛋白聚糖(SLRPs)为角膜的重要结构成分, 在角膜透明性的形成和维持方面发挥重要作用。SLRPs大量分布于角膜基质层中, 主要分为Ⅰ型、Ⅱ型和Ⅲ型。各成员之间存在补偿性和协同性作用, 共同调控基质层胶原纤维的形成和装配, 维持胶原纤维排列的高度有序性, 奠定角膜透明性的基础。Decorin和lumican分别是Ⅰ型和Ⅱ型SLRPs的主要功能成分, 二者基因表达改变或核心蛋白结构异常均会影响角膜基质层其他细胞外基质成分的正常含量和排列关系, 导致胶原纤维的形成、装配和排列异常, 造成角膜混浊。SLRPs可通过结合促纤维化细胞因子及其受体等调控角膜创伤修复和基质重塑, 为治疗角膜疾病和研究角膜透明性的分子机制提供依据。本文就SLRPs家族成员生物学活性及其在角膜透明性中的作用研究进展进行综述。     

细胞外基质对角膜创伤修复的作用

细胞外基质在角膜创伤修复过程中起至关重要的作用，其合成代谢受生长因子的调控，两者之间的关系是近年来令人感兴趣的新的研究领域，它将有助于阐明角膜创伤修复机制。本文概述了细胞外基质的成分、结构、功能和在角膜创伤修复中的作用以及生长因子在角膜创伤修复过程中对细胞外基质的调控。随着人们对角膜基础研究认识的不断深入，证明角膜细胞外基质（ECM）并非一种被动无活性的结构支架，而是一种活泼的动态物质，它影响着细胞的分化、增殖、粘附、形态发生等一系列生物学过程，是角膜细胞赖以传递与接受信息的物质基础，与许多角膜病变的生理病理过程密切相关。     

ECM,MMP与角膜结构和角膜损伤修复

细胞外基质是角膜的主要结构成分,赋予角膜特殊的生理功能。基质金属蛋白酶是调节细胞外基质降解代谢的主要酶类。本文概述了成熟角膜细胞外基质的组成及基质金属蛋白酶在角膜损伤修复过程中的作用。     

TGF-β在角膜损伤修复中的时间和空间分布

角膜损伤后的纤维化修复是角膜瘢痕形成的主要原因.转化生长因子-β(transforming growth factor-beta,TGF-β)在角膜的稳态平衡中起着至关重要的作用,是角膜损伤修复的重要参与者.同时,角膜上皮基底膜是角膜创伤修复过程中角膜上皮与基质相互作用的重要屏障.角膜损伤修复的不同阶段,各亚型TGF-β在角膜各种细胞及各个不同部位存在着分布差异,角膜上皮基底膜是否完整是影响该过程的重要因素.TGF-β不同亚型在时间和空间上的分布差异及变化与角膜的创伤修复过程中细胞的迁移、增殖、表型变化及细胞外基质沉着都紧密相关,是瘢痕愈合及无瘢痕愈合的细胞分子生物学基础.本文就TGF-β的生物学功能及其亚型在角膜损伤修复中的时间和空间分布情况作一综述.     

Growth factor, cytokine and protease interactions during corneal wound healing

Healing of corneal injuries is an exceptionally complex process involving the integrated actions of multiple growth factors, cytokines, and proteases produced by epithelial cells, stromal keratocytes, inflammatory cells, and lacrimal gland cells. Following corneal injury, basal epithelial cells migrate and proliferate in response to chemotactic cytokines and mitogenic growth factors, including epidermal growth factor and keratinocyte growth factor. Simultaneously, keratocytes adjacent to the injured area undergo apoptosis under the Fas/Fas ligand system, while more distant keratocytes transform into activated fibroblasts and migrate into the wound, where they begin synthesizing new extracellular matrix components that form the scar tissue under the dominant influence of the TGFb/ CTGF system. Epithelial cells and activated stromal fibroblasts also secrete growth factors and cytokines that have paracrine and autocrine functions. Corneal repair proceeds for the next several weeks to months, during which time the gene expression profile slowly returns to the pre-injury pattern and the provisional scar matrix slowly remodels by actions of matrix metalloproteinases. While minor epithelial injuries heal by regeneration of normal architecture, large stromal injuries heal by repair with irregular scar tissue that impairs the optical properties of the cornea.Also, if the integrated regulation of the wound healing process is interrupted at any point, the wound fails to heal properly and a corneal ulcer develops. Better understanding of the cellular and molecular changes that occur during repair of corneal wounds will provide the opportunity to design agents that selectively modulate key phases of corneal wound healing, resulting in scars that more closely resemble normal corneal architecture.     

Extracellular matrix and wound healing

Over the years, most researchers have approached corneal epithelial and stromal wound healing as separate events. However, it is becoming increasingly clear that even the simplest epithelial debridement wound results in keratocyte death and a subsequent stromal response to regenerate the affected area. Thus, the interaction between stromal and epithelial healing must be considered to fully understand corneal wound healing. Although wound healing has been an active area of research for many years, the advent of refractive surgery has stimulated research into the regulation of wound repair and provided important insights into the molecular components involved in repair. Epithelial and stromal wound healing are influenced by extracellular matrix components. The purpose of the current article is to review progress in the year 2000 toward understanding mechanisms involved in corneal wound healing and how extracellular matrix affects the healing processes.     

Corneal alkali burn in the rabbit. Waterbalance, healing and transparency.

Healing following a standardized central corneal alkali wound was studied morphologically in New Zealand white rabbits up to one year after the initial wound. Clinical examination, light and transmission electron microscopy was performed. The study was focused on how permanent scar tissue formed. Following the penetrating alkali injury, all cells (epithelium, keratocytes and endothelium) in the wound area disappeared. The fibroblasts/keratocytes repopulated an extensively swollen central corneal stroma. Cells and extracellular matrix filled stromal lacunae in an irregular fashion and upon deswelling the lacunae remained as irregularities in the stroma, reducing the transparency. In the periphery of the wound repopulation occurred in a less swollen stroma and normal cytoarchitecture and transparency resumed. It appears that the degree of swelling decides the degree of scar tissue formation in the corneal stroma following alkali wound healing.     

Corneal stromal wound healing: Major regulators and therapeutic targets

Corneal stromal wound healing is a complex event that occurs to restore the transparency of an injured cornea. It involves immediate apoptosis of keratocytes followed by their activation, proliferation, migration, and trans-differentiation to myofibroblasts. Myofibroblasts contract to close the wound and secrete extracellular matrix and proteinases to remodel it. Released proteinases may degenerate the basement membrane allowing an influx of cytokines from overlying epithelium. Immune cells infiltrate the wound to clear cellular debris and prevent infections. Gradually basement membrane regenerates, myofibroblasts and immune cells disappear, abnormal matrix is resorbed, and transparency of the cornea is restored. Often this cascade deregulates and corneal opacity results. Factors that prevent corneal opacity after an injury have always intrigued the researchers. They hold clinical relevance as they can guide the outcomes of corneal surgeries. Studies in the past have shed light on the role of various factors in stromal healing. TGFβ (transforming growth factor-beta) signaling is the central player guiding stromal responses. Other major regulators include myofibroblasts, basement membrane, collagen fibrils, small leucine-rich proteoglycans, biophysical cues, proteins derived from extracellular matrix, and membrane channels. The knowledge about their roles helped to develop novel therapies to prevent corneal opacity. This article reviews the role of major regulators that determine the outcome of stromal healing. It also discusses emerging therapies that modulate the role of these regulators to prevent stromal opacity.     

Corneal femtosecond laser keratotomy results in isolated stromal injury and favorable wound-healing response

PURPOSE: To examine the corneal repair response after intrastromal femtosecond (fs) laser keratotomy. METHODS: Twelve rabbits underwent monocular intrastromal keratotomy performed with an fs laser at a preoperatively determined corneal depth of 160 to 200 microm. The fs laser-induced corneal repair response was compared with that of nonoperated control eyes and eyes treated with photorefractive keratectomy (PRK). Follow-up examinations were performed 1, 3, 7, and 28 days after surgery. Corneas were evaluated using slit lamp, in vivo confocal microscopy, and light microscopy. The extracellular matrix components fibronectin and tenascin were located using immunofluorescence staining. Anti-Thy-1 and anti-alpha-SMA antibodies and phalloidin were used to identify repair fibroblasts. Cell proliferation and nuclear DNA fragmentation were detected using an anti-Ki-67 antibody and the TUNEL assay, respectively. RESULTS: Intrastromal fs keratotomy resulted in a hypocellular stromal scar discernible as a narrow band of increased reflectivity on slit lamp examination. Deposition of fibronectin and tenascin as well as death and subsequent proliferation of keratocytes were observed. No differentiation of keratocytes into Thy-1- or alpha-SMA-positive fibroblasts could be detected. In contrast, after PRK, which causes epithelial and stromal wounding, all markers for repair fibroblasts were found in subepithelial stromal layers. On slit lamp examination, a fibrotic scar and a corneal haze were revealed. CONCLUSIONS: Isolated stromal injury using an fs laser avoids epithelial injury and is associated with a favorable wound-healing response preserving corneal transparency. Thus, fs laser keratotomy is a highly selective laser treatment that can be useful for the treatment of refractive errors.     

Immunohistochemical study of subepithelial haze after phototherapeutic keratectomy

PURPOSE: Subepithelial haze is a frequent complication and is often the cause of regression after photorefractive keratectomy (PRK). The lack of understanding of this undesirable complication following PRK is in part due to the limited availability of suitable tissues for pathological studies. METHODS: We examined the expression of various extracellular components in the cornea of a 46-year-old man who underwent phototherapeutic keratectomy (PTK) to remove a central corneal scar secondary to trauma. The patient subsequently underwent penetrating keratoplasty. A scar-free region containing an area of slight subepithelial haze adjacent to normal cornea was used for immunohistochemical staining with antibodies directed against cytoskeletal proteins, ie, vimentin, desmin and smooth muscle actin, and the extracellular components, laminin, heparan sulfate, keratan sulfate, and collagen types III, IV, V, and VII. RESULTS: Immunohistochemistry revealed that basal epithelial cells expressed components of basement membrane. The stromal fibroblasts within the haze tissue were labeled by anti-smooth muscle actin antibodies, a characteristic of myofibroblasts, which synthesized and secreted extracellular matrix components that contributed to the formation of the disorganized collagenous matrix and may account for subepithelial haze. CONCLUSIONS: The expression patterns for the cytoskeletal proteins and extracellular components indicated that the formation of subepithelial haze is a process of tissue remodeling, involving both corneal basal epithelial cells and keratocytes during wound repair.     

准分子激光角膜切削术与准分子激光原位角膜磨镶术角膜创口愈合的对比实验研究

比较准分子激光角膜切削术(photorefractive keratectomy,PRK)与准分子激光原位角膜磨镶术(1aser in situ keratomileusis,LASIK)后激光对角膜组织的切削效应及角膜的愈合情况,从组织学角度探讨角膜雾状混浊(Haze)及屈光度数回退的成因。方法24只新西兰白兔按预矫屈光度数随机分为-4.00 D组和-8.00 D组,每只兔右眼行PRK,左眼行LASIK。术后10d,1、3及6个月观察Haze情况并验光,每组随机处死3只兔取角膜行光镜、电镜及免疫组化检查,检测胶原Ⅲ、胶原Ⅳ、纤维连结蛋白(fibronectine,FN)及转化生长因子-β(transforming growth factor-β1,TGF-β2)的含量。结果行PRK术的右眼术后出现不同程度的Haze及屈光度数回退,其程度与预矫正屈光度数成正比。行LASIK术的左眼术后除少数角膜瓣周围半环形混浊外,手术区域角膜透明,屈光度数回退较右眼轻。-4.00 D组右眼与左眼术后屈光度数均稳定,-8.00 D组右眼较左眼屈光度数回退明显。右眼术后角膜愈合反应重,恢复慢。6个月时角膜基质仍处于修复阶段。左眼术后除形成角膜上皮栓及对应处基质轻度增生外,手术区域角膜瓣与基质床间界面清晰,无明显增生,角膜基质愈合反应轻、恢复快。术后所有兔眼角膜均有TGF-β1表达及活化,持续时间与角膜愈合时间一致。Haze及屈光度数回退组织学改变为：角膜上皮细胞增生,基底膜不成熟,前基质角膜细胞活化、增殖,新生胶原Ⅲ合成、排列紊乱,细胞外基质FN在角膜上皮下沉积。结论LASIK矫正近视尤其是高度近视优于PRK;角膜伤口愈合特别是基质愈合的反应程度,是。Haze及屈光度数回退的关键;TGF-β1是角膜愈合过程中重要调节因子,可通过介导角膜上皮一基质相互作用,调节胶原Ⅲ及FN的含量,参与瘢痕形成。     

TGF-β2反义寡核苷酸对兔角膜基质创伤修复的影响

目的:探讨TGF-β2反义寡核苷酸对兔角膜基质成纤维细胞转化、增殖的作用,揭示其对角膜创伤修复的影响。方法:新西兰大白兔28只,双眼均制备角膜基质创伤模型,右眼为实验组,用浸有TGF-β2反义寡核苷酸的8-0薇乔缝线缝合角膜创口;左眼为对照组,用普通8-0薇乔缝线缝合角膜创口,分别于4,7,14,21d处死动物,取角膜后行免疫组化(α-SMA和PCNA)染色和电镜观察。结果:术后各时间段均发现,实验组α-SMA和PCNA阳性的成纤维细胞数明显少于对照组,但成纤维细胞的超微结构实验组和对照组比较无明显区别。结论:TGF-β2反义寡核苷酸可抑制兔角膜基质成纤维细胞转化、增殖,为调控角膜基质伤口修复提供了一个新的途径。     

Stromal-epithelial interactions in the cornea

Stromal-epithelial interactions are key determinants of corneal function. Bi-directional communications occur in a highly coordinated manner between these corneal tissues during normal development, homeostasis, and wound healing. The best characterized stromal to epithelial interactions in the cornea are mediated by the classical paracrine mediators hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF). HGF and KGF are produced by the keratocytes to regulate proliferation, motility, differentiation, and possibly other functions, of epithelial cells. Other cytokines produced by keratocytes may also contribute to these interactions. Epithelial to stromal interactions are mediated by cytokines, such as interleukin-1 (IL-1) and soluble Fas ligand, that are released by corneal epithelial cells in response to injury. Other, yet to be identified, cytokine systems may be released from the unwounded corneal epithelium to regulate keratocyte viability and function. IL-1 appears to be a master regulator of corneal wound healing that modulates functions such as matrix metalloproteinase production, HGF and KGF production, and apoptosis of keratocyte cells following injury. The Fas/Fas ligand system has been shown to contribute to the immune privileged status of the cornea. However, this cytokine-receptor system probably also modulates corneal cell apoptosis following infection by viruses such as herpes simplex and wounding. Pharmacologic control of stromal-epithelial interactions appears to offer the potential to regulate corneal wound healing and, possibly, treat corneal diseases in which these interactions have a central role.