间充质干细胞在角膜损伤修复和角膜移植中的应用

严重损伤后角膜的修复及角膜移植后的抗免疫排斥反应是角膜疾病治疗中的重点和难点.间充质干细胞可在特定环境下诱导分化为角膜上皮细胞、角膜基质细胞等细胞类型,或通过旁分泌作用抑制炎性反应及新生血管的生成,进而保护损伤组织,促进角膜修复.此外,间充质干细胞还具有抗炎和调节免疫的特性,使其在抗角膜移植排斥反应中发挥作用.     

Mesenchymal stromal cells for the treatment of ocular autoimmune diseases

Mesenchymal stromal cells, commonly referred to as MSCs, have emerged as a promising cell-based therapy for a range of autoimmune diseases thanks to several therapeutic advantages. Key among these are: 1) the ability to modulate innate and adaptive immune responses and to promote tissue regeneration, 2) the ease of their isolation from readily accessible tissues and expansion at scale in culture, 3) their low immunogenicity enabling use as an allogeneic “off-the-shelf” product, and 4) MSC therapy's safety and feasibility in humans, as demonstrated in more than one thousand clinical trials. Evidence from preclinical studies and early clinical trials indicate the therapeutic potential of MSCs and their derivatives for efficacy in ocular autoimmune diseases such as autoimmune uveoretinitis and Sjögren's syndrome-related dry eye disease. In this review, we provide an overview of the current understanding of the therapeutic mechanisms of MSCs, and summarize the results from preclinical and clinical studies that have used MSCs or their derivatives for the treatment of ocular autoimmune diseases. We also discuss the challenges to the successful clinical application of MSC therapy, and suggest strategies for overcoming them.     

角膜神经调控眼表微环境的研究进展

角膜是眼前段透明的外层结构，由高密度的神经组织支配。在角膜神经支配过程中，三叉神经节起源的角膜神经穿过上皮层和基质层中不同类型的角膜细胞。角膜基质细胞、上皮细胞、免疫细胞等多种细胞和角膜神经之间发生密切的相互作用，共同维持角膜微环境稳态。此外，角膜神经参与许多眼表疾病的发生发展过程。角膜神经释放多种活性肽物质，参与调控角膜感觉、维持上皮完整性和增殖、促进伤口愈合及调控角膜局部炎症和免疫反应等。本文对角膜神经在眼表微环境调控作用的研究进展进行综述，为角膜神经相关疾病的研究及治疗提供新的思路。     

大鼠骨髓间充质干细胞体外可诱导分化为角膜上皮细胞

目的:探讨骨髓间充质干细胞 (mesenchymal stem cell,MSC) 分化为角膜上皮细胞的可塑性及其重建角膜上皮的可能性.方法:用密度梯度离心法结合贴壁培养法分离纯化大鼠骨髓MSC,经体外与角膜基质细胞共培养诱导分化,免疫荧光法检测角膜上皮细胞特异标志物K12的表达.结果:体外培养的大鼠骨髓MSC表现出很强的增殖潜能,原代培养的骨髓MSC CD29免疫荧光染色阳性,CD34和CD45为阴性,符合骨髓MSC的特征.MSC与角膜基质细胞共培养1wk后大部分细胞分化为间质细胞,少部分细胞形态上相对偏小,免疫荧光检测这部分细胞表达角膜上皮细胞特异性标志角蛋白K12.结论:体外培养的MSC在角膜基质细胞的诱导下可横向分化为角膜上皮细胞.     

人角膜基质细胞诱导人骨髓间充质干细胞分化为角膜上皮细胞的初步研究

目的 探讨人骨髓间充质干细胞（MSC）体外分化为角膜上皮细胞的可行性.方法 实验研究.分别取第3代人MSC和自行培养的第3代人角膜基质细胞共同培养1周,实验组在Transwell共培养体系中培养,对照组不放置Transwell小室培养.1周后,观察实验组和对照组中人MSC光镜特征、间接免疫细胞化学染色和电镜结构,对被诱导分化的细胞进行综合鉴别.结果 第3代人MSC和人角膜基质细胞在体外培养条件下均能够较快贴壁生长.两种细胞共同培养1周后,可见部分细胞形态上呈上皮细胞特征,单克隆抗体AE1染色呈阳性,电镜下可见微绒毛、桥粒和张力丝等典型上皮细胞结构特征.结论 体外培养的人MSC在人角膜基质细胞的诱导下可能会分化为人角膜上皮样细胞.     

Neuroimmune crosstalk in the cornea: The role of immune cells in corneal nerve maintenance during homeostasis and inflammation

In the cornea, resident immune cells are in close proximity to sensory nerves, consistent with their important roles in the maintenance of nerves in both homeostasis and inflammation. Using in vivo confocal microscopy in humans, and ex vivo immunostaining and fluorescent reporter mice to visualize corneal sensory nerves and immune cells, remarkable progress has been made to advance our understanding of the physical and functional interactions between corneal nerves and immune cells. In this review, we summarize and discuss recent studies relating to corneal immune cells and sensory nerves, and their interactions in health and disease. In particular, we consider how disrupted corneal nerve axons can induce immune cell activity, including in dendritic cells, macrophages and other infiltrating cells, directly and/or indirectly by releasing neuropeptides such as substance P and calcitonin gene-related peptide. We summarize growing evidence that the role of corneal intraepithelial immune cells is likely different in corneal wound healing versus other inflammatory-dominated conditions. The role of different types of macrophages is also discussed, including how stromal macrophages with anti-inflammatory phenotypes communicate with corneal nerves to provide neuroprotection, while macrophages with pro-inflammatory phenotypes, along with other infiltrating cells including neutrophils and CD4⁺ T cells, can be inhibitory to corneal re-innervation. Finally, this review considers the bidirectional interactions between corneal immune cells and corneal nerves, and how leveraging this interaction could represent a potential therapeutic approach for corneal neuropathy.     

间充质干细胞治疗干眼的研究进展

间充质干细胞(mesenchymal stem cells,MSCs)是一种具有全能干细胞特点来源于中胚层发育的早期细胞,具有多向分化的潜能,在特定的诱导条件下可分化为多种细胞.目前,随着MSCs在治疗自身免疫性疾病方面研究日渐深入,越来越多的学者致力于MSCs对干眼的免疫治疗作用的研究.干眼发病率逐年增高,免疫相关性干眼治疗期长,且效果欠佳,影响人们生活质量,迫切需要开发新型治疗方法.因此本文主要就MSCs对干眼治疗方面做系统的综述.     

持续性角膜上皮缺损的治疗进展

持续性角膜上皮缺损(PED/PCEDs)是指角膜损伤后10～14d内,在接受了相应治疗后,角膜也未能迅速重新形成上皮并闭合而导致的一种角膜疾病。角膜上皮的破坏和基质层的损伤容易使眼部受到感染、发生基质溃疡、穿孔、瘢痕,甚至丧失视力。就目前而言,临床医生对PED的治疗仍然面临相当大的挑战。标准的治疗方法包括配戴绷带隐形眼镜和使用人工泪液治疗,而新开发的药物则可以通过促进各类生长因子的生成使角膜重新形成上皮,进一步配合相应外科手术为角膜提供神经支配,以此达到治疗的效果。此外,确诊PED后应尽早接受治疗,以避免继发性并发症。本文就PED的流行病学、病因学、诊断与临床表现、治疗方法及预后进行综述。     

间充质干细胞对自身免疫性干眼的免疫调控作用研究进展

间充质干细胞(mesonchymal stem cells,MSCs)具有良好的免疫调节功能,可以抑制多种免疫细胞群增殖,直接作用抑制T淋巴细胞的活化增殖,上调Treg细胞或通过分泌可溶性因子调节Th1/Th2反应平衡而发挥免疫调节作用,通过抗炎、调控各类性细胞因子的表达等方面抑制炎性因子的分泌同时促进抑炎因子分泌,降低基质金属蛋白酶2(MMP-2)、MMP-9等可能促进泪腺组织损害因子的表达水平,进而发挥其免疫调节作用,MSCs能减轻自身免疫性干眼的临床指标,恢复部分泪腺组织的分泌功能,本文就MSCs对自身免疫性干眼免疫调控的研究进展进行综述.     

Keratoplasty after mustard gas injury: clinical outcome and histology

PURPOSE: Ocular injury by mustard gas can lead to severe eye damage with a delayed course. We report the corneal histology and follow-up after keratoplasty in a patient with mustard gas injury. METHODS: The patient presented with recurrent painful corneal inflammation in both eyes not improving under local therapy. Visual acuity impaired to handmovements. A penetrating keratoplasty was performed on the left eye and afterwards an autorotation keratoplasty on the right eye with a later corneal graft. RESULTS: After the operation of the left eye the patient was immediately painfree and the visual acuity improved to 0,4. So far there have been no signs for transplant rejection or inflammation. Histology of the cornea revealed massive stromal necrosis, and signs of chronic inflammation. Despite denervation of the cornea after autorotation keratoplasty the right eye was still painful and became only painfree after corneal transplantation. CONCLUSION: There has been not much experience with corneal transplantation after mustard gas injury and there is a high risk for transplant rejection due to inflammation and vascularisation of the cornea. Successful and painfree healing with keratoplasty seems only possible after complete removal of the necrotic material.     

Use of amniotic membrane graft and corneal transplantation in a patient with bilateral keratomalacia induced by uncontrolled phenylketonuria

PURPOSE: To report a patient with a rare complication of bilateral keratomalacia induced by uncontrolled phenylketonuria (PKU) that was successfully treated with amniotic membrane transplantation in 1 eye and penetrating keratoplasty in the second eye. METHODS: Case report and literature review. RESULTS: A 9-month-old girl with uncontrolled PKU was referred to our clinic because of bilateral keratomalacia. Slit-lamp examination of the right eye revealed 2 large corneal erosions with stromal thinning on the nasal and inferior regions of the right cornea. Left eye examination revealed a large area of melting involving two thirds of the cornea with corneal perforation and iris bulging on the temporal side and no anterior chamber. She underwent amniotic membrane transplantation on her right cornea and penetrating keratoplasty on her left cornea. Treatment after surgery included antibiotic and steroid eye drops and a special diet regimen with partial phenylalanine intake. Examination under anesthesia 4 months after surgery revealed intact cornea in her right eye and a clear corneal graft with a deep anterior chamber on her left eye. Intraocular pressure was normal in both eyes. CONCLUSIONS: Bilateral keratomalacia, although a rare ophthalmic manifestation of PKU, can cause a severe corneal injury that may threaten the eyeball integrity. Surgical treatments with amniotic membrane graft and corneal transplantation, along with the appropriate diet treatment, were found to be effective procedures, yielding rapid healing of the corneal surface.     

Improvement of HSV-1 necrotizing keratitis with amniotic membrane transplantation

PURPOSE: Stromal herpes simplex virus keratitis (HSK) is an immune-mediated disease. Previous studies have indicated that T cells, neutrophils, and macrophages contribute to the tissue damage in HSK. It has been shown that human amniotic membrane promotes epithelial wound healing and has diverse anti-inflammatory effects. In this study, the effect of amniotic membrane transplantation (AMT) on corneal wound healing and on inflammation in mice with necrotizing HSK was examined. METHODS: BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain). In 16 mice that exhibited severe ulcerating HSK, the cornea was covered with a preserved human amniotic membrane as a patch. Corneas in 16 infected mice remained uncovered and served as a control. On days 2 and 7 after surgery, the amniotic membrane was removed (eight mice in each group), the HSV-1-infected cornea was evaluated clinically, and the eye was enucleated. Tissue sections were analyzed histologically for epithelialization and cellular infiltration and immunohistochemically with anti-CD3 mAb to T cells, anti-CD11b mAb to both macrophages and neutrophils, or anti-F4/80 mAb to macrophages. RESULTS: Profound regression of corneal inflammation and rapid closure of epithelial defects were observed clinically within 2 days in the amniotic membrane-covered eyes, whereas HSV-1 keratitis and ulceration progressed in all mice in the control group (P < 0.001). Histologically, corneal edema and inflammatory infiltration, and immunohistochemically the number of CD3(+), CD11b(+), and F4/80(+) cells in the cornea were markedly decreased at 2 and 7 days after amniotic membrane application, compared with the uncovered control corneas (P < 0.001). CONCLUSIONS: AMT promotes rapid epithelialization and reduces stromal inflammation and ulceration in HSV-1 keratitis. AMT in mice with HSV necrotizing stromal keratitis appears to be a useful model for investigating the effect and the action mechanism of human amniotic membrane.     

Supramolecular host-guest hyaluronic acid hydrogels enhance corneal wound healing through dynamic spatiotemporal effects

Severe corneal wounds can lead to ulceration and scarring if not promptly and adequately treated. Hyaluronic acid (HA) has been investigated for the treatment of corneal wounds due to its remarkable biocompatibility, transparency and mucoadhesive properties. However, linear HA has low retention time on the cornea while many chemical moieties used to crosslink HA can cause toxicity, which limits their clinical ocular applications. Here, we used supramolecular non-covalent host-guest interactions between HA-cyclodextrin and HA-adamantane to form shear-thinning HA hydrogels and evaluated their impact on corneal wound healing. Supramolecular HA hydrogels facilitated adhesion and spreading of encapsulated human corneal epithelial cells ex vivo and improved corneal wound healing in vivo as an in situ-formed, acellular therapeutic membrane. The HA hydrogels were absorbed within the corneal stroma over time, modulated mesenchymal cornea stromal cell secretome production, reduced cellularity and inflammation of the anterior stroma, and significantly mitigated corneal edema compared to treatment with linear HA and untreated control eyes. Taken together, our results demonstrate supramolecular HA hydrogels as a promising and versatile biomaterial platform for corneal wound healing.     

Tandem-scanning (confocal) microscopy of the full-thickness cornea

We have utilized a radically new type of optical scanning microscope to study the full-thickness morphology of the intact cornea in an excised human eye bank eye and in freshly sacrificed rabbit eyes in situ. This technology enables one to study corneal morphology layer by layer in extremely thin sections, only disturbing the tissue with an applanating tip. We have demonstrated the cells of the corneal surface, subsurface cells, the topography of Bowman's membrane, corneal lamellae, stromal keratocytes, and the corneal endothelium. The application of this technology lends itself to the in vivo examination of the human cornea. This should aid us greatly in the study of normal morphology, disease states, and the reaction of the cornea in wound healing.     

Use of nerve growth factor to treat congenital neurotrophic corneal ulceration

PURPOSE: This study was designed to assess the efficacy of nerve growth factor in the treatment of neurotrophic corneal ulceration in a child with bilateral congenital corneal anesthesia secondary to trigeminal insufficiency. METHODS: A 5-month-old child presented to the casualty department with a 2-week history of red eyes and right corneal ulceration. Slit-lamp examination showed a central defect in the right corneal epithelium with underlying stromal opacification, only mild conjunctival inflammation with slight decreased tear production, and otherwise apparently normal eyes. Initially this was investigated as an infected ulcer and treated for several weeks as herpetic ulceration with no beneficial effect. Further clinical examination demonstrated bilateral decreased corneal sensation along with decreased facial sensation in keeping with congenital trigeminal nerve insufficiency. Investigation with magnetic resonance imaging showed no obvious abnormality. Conservative treatment with lubricants resulted in progressive right corneal stromal loss, and no healing occurred in the left corneal ulcer. Bilateral large lateral tarsorrhaphies were performed. Despite this, the left corneal ulcer demonstrated no improvement and increasing stromal opacification was noted. Topical nerve growth factor (NGF) was then used to treat the left cornea and resulted in epithelial healing within 1 week. Treatment was continued for a further 10 days after epithelial healing. Despite conventional treatment on 3 separate occasions, further epithelial breakdown occurred. Topical NGF treatment resulted in a rapid improvement and healing of the epithelial defect.'At present, the patient is receiving a 6-month continuous treatment plan of NGF. RESULTS: Persistent epithelial defects (PED) secondary to neurotrophic ulceration have responded to topical NGF on 3 separate occasions during a 2-year period. The corneal epithelium now remains intact, and the cornea has no vascularization; however, mild anterior stromal opacification has gradually increased despite prolonged NGF treatment. CONCLUSION: NGF seems to represent a safe and efficacious treatment option to restore the integrity of corneal epithelium in which there is congenital corneal anesthesia because of trigeminal insufficiency. However, this treatment alone is insufficient to prevent progressive anterior stromal opacification.     

Mesenchymal stromal cells for corneal transplantation: Literature review and suggestions for successful clinical trials

Corneal transplantation is a routine procedure for patients with corneal blindness. Despite the streamlining of surgical techniques and deeper understanding of the cellular and molecular pathways mediating rejection, corticosteroids are still the main immunosuppressive regimen in corneal transplantation, and the 15-year survival of corneal transplants remains as low as 50%, which is poorer than that for most solid organ transplants. Recently, mesenchymal stromal cells (MSCs) with unique regenerative and immune-modulating properties have emerged as a promising cell therapy to promote transplant tolerance, minimize the use of immunosuppressants, and prevent chronic rejection. Here, we review the literature on preclinical studies of MSCs for corneal transplantation and summarize the key findings from clinical trials with MSCs in solid organ transplantation. Finally, we highlight current issues and challenges regarding MSC therapies and suggest strategies for safe and effective MSC-based therapies in clinical transplantation.     

Progress in corneal wound healing

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β (TGF-β) system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells.     

The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells

The corneal wound healing cascade is complex and involves stromal-epithelial and stromal-epithelial-immune interactions mediated by cytokines. Interleukin-1 appears to be a master modulator of many of the events involved in this cascade. Keratocyte apoptosis is the earliest stromal event noted following epithelial injury and remains a likely target for modulation of the overall wound healing response. Other processes such as epithelial mitosis and migration, stromal cell necrosis, keratocyte proliferation, myofibroblast generation, collagen deposition, and inflammatory cell infiltration contribute to the wound healing cascade and are also likely modulated by cytokines derived from corneal cells, the lacrimal gland, and possibly immune cells. Many questions remain regarding the origin and fate of different cell types that contribute to stromal wound healing. Over a period of months to years the cornea returns to a state similar to that found in the unwounded normal cornea.     

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.     

角膜伤口愈合机制对准分子激光角膜屈光手术的启示

角膜伤口愈合反应是由细胞因子、生长因子、趋化因子介导的复杂级联反应。角膜上皮细胞和基底膜在这一过程中起了重要的作用,而这二者在准分子激光角膜屈光手术中常常被损伤。因此,深刻理解角膜伤口愈合过程以及并发症产生的原因,对提高准分子激光角膜屈光手术的有效性和安全性具有重要意义。就角膜伤口愈合过程及其机制进行综述。