角膜缘微环境细胞的研究进展

角膜缘微环境细胞（limbal niche cell,LNC）是近年来发现的一种来自于角膜缘干细胞微环境且具有血管内皮祖细胞和间充质干细胞特性的多能干细胞。LNC具有分化为血管内皮细胞、角膜上皮细胞、角膜基质细胞等多种细胞的潜能。体外实验发现,LNC在三维立体培养环境中可以维持角膜缘干细胞的特性;动物实验发现,LNC可以预防和治疗碱烧伤所致的角膜缘干细胞缺乏。LNC还可以促进角膜基质的无瘢痕修复,减少修复过程中的新生血管化,提示其可以重建角膜基质。因此,在角膜缘干细胞缺乏、角膜基质损伤等疾病的治疗中,LNC可能是一种理想的治疗细胞。     

角膜上皮干细胞定位特征的免疫组织化学研究

利用单克隆抗体ＡＥ５与分化型角膜上皮细胞中角蛋白Ｋ３特异性结合,研究缺乏分化标志特征的角膜上皮干细胞定位特点,应用免疫组织化学方法显示Ｋ３阳性表达的区域分布于除角膜缘上皮基底部以外所有角膜上皮细胞中,角膜上皮干细胞存在于角膜缘基底部ＡＥ５抗体反应阴性细胞中,即角膜干细胞位于角膜缘上皮层基底部。     

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.     

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.     

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.     

Regeneration of rabbit cornea following excimer laser photorefractive keratectomy: a study on gap junctions, epithelial junctions and epidermal growth factor receptor expression in correlation with cell proliferation

Corneal wound repair was investigated in rabbits following excimer laser ablation of a 6 mm diameter and 90 microm deep disc. In the healing process particular attention was focused on the epithelium where gap junction expression and the rearrangement of desmosomes and hemidesmosomes were correlated with cell proliferation and epidermal growth factor receptor expression. Immunofluorescence-based confocal laser scanning microscopy, semithin resin section morphology and electron microscopy were utilized. In resting cornea two isotypes of gap junctions, confined to different regions in the same basal epithelial cells, were detected. Particulate connexin43 (alpha1) immunostaining was concentrated on the apical while the connexin26 type (beta2) in the baso-lateral cell membranes. This is the first report of connexin26 in the cornea. Connexin43 was found also in corneal keratocytes and endothelial cell. Since the two connexins do not form functioning heteromeric channels and have selective permeabilities they may serve alternative pathways for direct cell-cell communication in the basal cell layer. During regeneration both connexins were expressed throughout the corneal epithelium including the migrating cells. They also showed transient up-regulation 24 hr after wounding in the form of overlapping relocation to the upper cell layers. At this time, basal epithelial cells at the limbal region, adjacent to the wound and those migrating over the wounded area all expressed membrane bound epidermal growth factor receptor and they were highly proliferating. In conclusion, like in other stratified epithelia connexin26 is also expressed in the cornea. Transient up-regulation and relocation of connexins within the regenerating epithelium may reflect the involvement of direct cell-cell communication in corneal wound healing. Mitotic activity in the migrating corneal epithelial cells is also a novel finding which is probably the sign of the excessive demand for new epithelial cells in larger wounds not met alone by the proliferating limbal stock.     

Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6+/- mouse cornea

PURPOSE: Corneal wound healing involves a cascade of interactions between the epithelium and stroma. Pax6 is upregulated, and early events include epithelial cell migration and apoptosis of superficial keratocytes. The mouse heterozygous Pax6 (Pax6+/-) corneal phenotype mimics human aniridia-related keratopathy (ARK), and some aspects of wound healing have been shown to be abnormal, including matrix metalloproteinase (MMP)-9 expression. The purpose of this study was to test whether the Pax6+/- genotype affects corneal wound-healing responses, including stromal cell apoptosis, epithelial cell migration rate, and MMP secretion in culture. METHOD: Pax6+/- and wild-type (Pax6+/+) mice were killed and their corneas wounded by epithelial debridement. Whole eyes were cultured in organ culture and corneal epithelial healing rates and keratocyte apoptosis were quantified by topical fluorescein staining and TUNEL, respectively. Dissociated corneal epithelial cells from Pax6+/- and wild-type mice were cultured, and the activities of secreted MMP-9 were determined by zymography. RESULTS: Wound-healing rates during the first 6 hours were significantly faster for larger wounds and for Pax6+/- corneas. Compared with wild-type, wounded Pax6+/- eyes showed significantly more stromal cell apoptosis, and cultured Pax6+/- corneal epithelial cells produced lower MMP-9 activity. CONCLUSIONS: The cumulative effect of abnormal wound-healing responses, characterized by increased stromal cell apoptosis and reduced levels of MMP-9 secretion may contribute to the corneal changes in the Pax6+/- mice. Possible contributions of elevated stromal cell apoptosis and other abnormal wound-healing responses to ARK are discussed.     

Corneal cells: chatty in development,homeostasis, wound healing,and disease

PURPOSE: To provide an overview of cell-cell interactions in the cornea that have a critical role in corneal development, homeostasis, wound healing, and disease. DESIGN: Review. METHODS: Review of the literature. RESULTS; Cell-cell interactions make critical contributions to development, homeostasis, and wound healing in the cornea. Many of these interactions are mediated by cytokines, growth factors, and chemokines. The best characterized are stromal-epithelial interactions between epithelial cells and stromal cells such as keratocytes, keratoblasts, and myofibroblasts. However, interactions also occur between corneal nerves and epithelial cells and between corneal cells (epithelial cells and stromal cells) and corneal immune cells. Although investigations are limited, it is likely that there are interactions between corneal endothelial cells and keratocytes in the posterior stroma. CONCLUSIONS: Cellular communications in the cornea are critical during development, homeostasis, and wound healing. Disorders of cellular communication likely contribute to many corneal diseases.     

利用组织工程方法治疗角膜缘干细胞缺失的相关研究进展

角膜缘干细胞在角膜上皮更新和创伤愈合中起重要作用,角膜缘干细胞缺失可导致各种严重眼表面疾患。因为治疗上存在自体移植的健眼损伤问题,同种异体移植的排斥反应以及供体不足等问题,近年来随着对干细胞研究的深入以及组织工程学的发展,有利于克服和解决上述问题,为角膜缘干细胞缺乏的治疗带来了曙光。本文通过近年来组织工程治疗角膜缘干细胞缺失的相关研究的回顾,归纳了种子细胞和支架材料选择两方面的研究进展,以探索该领域存在的问题和未来的研究方向。（国际眼科纵览, 2017,  41:   346-351）     

我国角膜基础和临床研究的现状及发展

现已公认,角膜上皮干细胞存在于角膜缘基底层.近来研究发现,除角膜缘外,在角膜上皮的基底层细胞还存在着寡能干细胞,同样可以参与上皮细胞的损伤修复.角膜内皮细胞正常在体内不能再生,近来研究发现内皮细胞在特定部位也存在有干细胞,在特定条件下可以转化为内皮细胞.角膜基质细胞同样存在干细胞,并且基质细胞具有异质性,在特定环境下可以表现其神经嵴前体细胞的特性,是HSV-1潜伏感染的基础.真菌性角膜炎不同致病菌种的菌丝在角膜内有不同的生长方式的发现,对推动真菌性角膜炎的手术治疗起了极为重要的作用.我国棘阿米巴角膜炎的主要危险因素是由植物和泥土外伤造成,与国外报道的角膜接触镜是主要危险因素不同,为临床的诊断提供了新的重要信息.在此,将近5年角膜的基础和临床研究进展进行分析.     

Corneal Stem Cells: Bridging the Knowledge Gap

Stem cell research offers hope to countless patients whose conditions have heretofore been deemed incurable. Better understanding of stem cell behaviors and functions will lead to insights into biological mysteries encompassing the fields of angiogenesis, development, tissue homeostasis, wound healing, and carcinogenesis. Clarity of vision requires smooth ocular surface on which the corneal epithelial cells undergo continuous turnover every 3 to 10 days. Tragically, many patients are blinded and devastated by severe ocular surface diseases due to limbal stem cell deficiency even though, besides opaque cornea, their eyes are otherwise healthy. Corneal stem cell transplantation offers hope by creating clear windows for these eyes; unfortunately, the long-term successful outcome remains limited. The nature of corneal epithelial stem cell is poorly understood, but many circumstantial evidences suggest the presence of “source cells” in the limbal region of the eye. Nonetheless, the precise biomarker of corneal stem cell remains elusive. The stem cell puzzle can be solved with application of the fundamental scientific method—asking salient questions at the right time and finding answers using keen observations and proper tools. Readily accessibility and structural simplicity of the cornea lend themselves to study of the stem cell biology. The ability to identify and isolate corneal stem cell will be a gateway to meaningful investigation into its biology. This advance will also have direct impact on improving the efficacy of promising stem-cell-based therapies, including limbal stem cell transplantation.     

Corneal stem cells: bridging the knowledge gap

Stem cell research offers hope to countless patients whose conditions have heretofore been deemed incurable. Better understanding of stem cell behaviors and functions will lead to insights into biological mysteries encompassing the fields of angiogenesis, development, tissue homeostasis, wound healing, and carcinogenesis. Clarity of vision requires smooth ocular surface on which the corneal epithelial cells undergo continuous turnover every 3 to 10 days. Tragically, many patients are blinded and devastated by severe ocular surface diseases due to limbal stem cell deficiency even though, besides opaque cornea, their eyes are otherwise healthy. Corneal stem cell transplantation offers hope by creating clear windows for these eyes; unfortunately, the long-term successful outcome remains limited. The nature of corneal epithelial stem cell is poorly understood, but many circumstantial evidences suggest the presence of "source cells" in the limbal region of the eye. Nonetheless, the precise biomarker of corneal stem cell remains elusive. The stem cell puzzle can be solved with application of the fundamental scientific method-asking salient questions at the right time and finding answers using keen observations and proper tools. Readily accessibility and structural simplicity of the cornea lend themselves to study of the stem cell biology. The ability to identify and isolate corneal stem cell will be a gateway to meaningful investigation into its biology. This advance will also have direct impact on improving the efficacy of promising stem-cell-based therapies, including limbal stem cell transplantation.     

角膜缘部干细胞对角膜上皮创伤愈合的影响

利用角膜缘上皮移植，结膜移植和非手术的方法分别对角膜上皮创伤伴随膜缘损伤的兔眼进行实验研究，结果表明角膜缘上皮移植组上皮愈合时间短（平均８．５天），且无杯状细胞，无或极少量新生血管；结膜移植组，上皮愈合时间延长（平均１１．５天），含有杯状细胞和新生血管，非手术组形成角膜血管翳性混浊。而不件随角膜缘损伤仅有角膜中央皮皮创伤的兔眼愈合时间最短（平均３．５天），愈合后无新生血管及怀状细胞。结果证实角膜部     

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。     

培养角膜缘干细胞羊膜移植治疗碱烧伤动物的实验研究

目的 观察培养生长于羊膜的角膜缘干细胞移植的治疗角膜缘碱烧伤伤的效果。方法 将兔角膜缘干细胞在的代培养后接种于羊膜,对新西兰大白兔角膜缘碱烧伤动物模型行角膜缘干细胞羊膜移植术,并对治疗后的角膜进行临床及病理学检查。结果 体外培养的兔角膜缘士细胞可在羊膜上继续增殖、分化为密集的角膜上皮细胞层;角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮完整基质细胞浸润减轻、新生血管减少。组织病理学染色证实,角膜缘     

Construction of a complete rabbit cornea substitute using a fibrin-agarose scaffold

PURPOSE: To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering. METHODS: Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy. RESULTS: All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium. CONCLUSIONS: These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.     

Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle

Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells.     

Limbal Stem Cell Transplantation and Complications

ABSTRACT

Corneal epithelial stem cells are adult somatic stem cells located at the limbus and represent the ultimate source of transparent corneal epithelium. When these limbal stem cells become dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. LSCD is a major cause of corneal scarring and is particularly prevalent in chemical and thermal burns of the ocular surface. LSCD leads to conjunctivalization of the corneal surface, neovascularization, recurrent or persistent epithelial defects, ocular surface inflammation, and scarring that, in turn, lead to decreased vision, pain, and impaired quality of life. Several techniques have been reported for limbal stem cell transplantation (LSCT). We introduce the surgical techniques, examine the success rate, and discuss the postoperative complications of conjunctival limbal autograft (CLAU), cultivated limbal stem cell transplantation (CLET), simple limbal epithelial transplantation (SLET), and limbal allograft, including keratolimbal allografts (KLAL) and living-related conjunctival allograft (LR-CLAL).