细胞凋亡与角膜疾病

Doh1man等 [1 ] 30年以前发现 ,前基质层的角膜细胞在上皮损伤后消失 ,当时被认为是由于机械损伤引起的组织暴露于周围环境及渗透压改变引起的。Cam pos等 [2 ] 于 90年代初在这一研究基础上 ,发现上皮损伤后角膜细胞的消失是由细胞程序性死亡 (PCD)即凋亡引起的。凋亡是生物体在进化过程中去除不需要的细胞 ,而同时不释放降解酶引起周围组织细胞损伤的自身调节过程。凋亡与机体发育、体内环境稳定、感染、损伤和疾病的病理生理变化都密切相关。1　细胞凋亡与大泡性角膜病变大泡性角膜病变时 ,角膜细胞经常破坏并穿过 Bowman膜 ,形成上皮…     

角膜基质细胞研究进展

角膜基质细胞，存在于角膜基质层中，在正常情况下，处于静止状态。但当角膜损伤时，不同上皮来源的因子及环境信号，将影响角膜基质细胞的应答反应，决定着角膜能否完全被修复或形成角膜瘢痕。本文就角膜基质细胞的分布、形态、受激后的应答反应情况及与角膜疾病的关系作一综述。     

准分子激光原位角膜磨镶术后角膜细胞凋亡的研究

目的 了解准分子激光原位角膜磨镶术（laser in situ keratomileusis,LASIK）后早期角膜基质细胞凋亡的特点并探索LASIK术后角膜愈合反应程度与角膜刀的机械损伤、激光能量的相关性。方法 20只新西兰白兔,其中10只兔左眼为LASIK-4.0D组、右眼为LASIK-8.0D组、另10只兔左眼为单纯角膜瓣组、右眼作为空白对照组,在术后4小时处死兔取角膜制作病理切片,行透射电镜检查,用脱氧核糖核苷酸末端转移酶介导的缺口末端标法（terminal-deoxynucleotidyl transferase medi-ated dUTP nick end labeling,TUNEL）原位显示凋亡细胞,激光扫描共聚焦显微镜观察凋亡细胞形态,定量统计比较凋亡水平差别。结果 三个手术组中角膜基质中均出现凋亡细胞,主要分布于角膜瓣和基质床交接面两侧50μm范围内的基质层中;瓣缘的凋亡水平高于中央部。而空白对照组中,凋亡细胞仅出现在表层上皮,角膜基质细胞未见凋亡。三个手术组的TUNEL阳性细胞计数进行方差分析,三组之间差异无显著意义（F=1.008,P〉0.05）;每组不同个体的TUNEL阳性细胞计数存在明显的差异。结论 正常角膜基质细胞不发生凋亡。角膜基质细胞凋亡与角膜损伤有关。LASIK术后早期即可出现角膜基质细胞的凋亡表达,该表达与角膜瓣的制作有关,与激光切削的深度、能量无明显的相关性。     

Bad在正常人角膜上皮细胞中表达的免疫组化研究

目的 探讨正常人角膜上皮中Bad蛋白的表达状况。方法 用免疫组化的方法（SP）观察8例Bad在正常成人角膜组织中的表达状态。结果 正常人角膜上皮表层细胞、角膜上皮基底细胞，包括角膜缘基底细胞中均有Bad表达，中央及周边部角膜上皮表层细胞Bad表达量无明显区别，角膜上皮表层和基底细胞间的绝大多数有丝分裂后仅部分细菌有Bad表达。结论 Bad参与了正常角膜上皮细胞的自我更新过程，可能具有促进角膜上皮细胞凋亡的作用。如能抑制Bad基因的表达可能会在一定程度上延缓角膜上皮的脱落，加速角膜上皮损伤后的修复及维持正常角膜上皮的完整与稳定。     

去除上皮瓣的微型角膜刀法准分子激光角膜上皮瓣下磨镶术后早期兔角膜的修复方式研究

目的 通过对兔微型角膜刀法准分子激光角膜上皮瓣下磨镶术(Epi-LASIK)和去除上皮瓣的(Flap-free)Epi-LASIK术后早期角膜组织的研究,探讨两种手术方式各自在角膜愈合方式方面的特点.方法 实验研究.27只实验兔,24只按不同取材时间1、2、4、7 d采用随机数字表法分为4组,每组6只兔,一只眼行Epi-LASIK,另一只眼行Flap-free Epi-LASIK手术.另3只兔作为空白对照组.用VISX20/20准分子激光仪作-10.00 D切削,另3只兔作为空白对照,通过观察术后1、2、47 d角膜上皮愈合方式、检测角膜基质细胞数量、碱性成纤维细胞生长因子(bFGF)、角膜基质细胞凋亡的表达来探讨Flap-free Epi-LASIK、Epi-LASIK两种术式术后早期角膜创伤愈合方式的差别.对Epi-LASIK和Flap-free Epi-LASIK术后角膜基质bFGF表达、细胞数量变化和细胞凋亡情况的数据采用t检验进行统计学处理.结果 通过光镜和透射电镜观察发现,Flap-free Epi-LASIK术后第1天有1层新生角膜上皮覆盖切削区域,以后逐层愈合.Epi-LASIK术后第1天角膜上皮基底膜与基质贴附较为紧密,但是在第2天可以看到有角膜上皮瓣水肿发生.术后4 d Flap-free Epi-LASIK角膜基质细胞数量分别为(33.85±3.39)个,多于Epi-LASIK组(t=-2.315,P<0.05).术后2 d、4 d组Flap-free Epi-LASIK角膜基质bFGF表达量分别为101.75±8.07和110.56±9.00,高于Epi-LASIK(t=-2.339,-2.396;P<0.05).术后2 d、4 d组Flap-free Epi-LASIK基质细胞凋亡数量也少于Epi-LASIK(t=3.332,2.989;P<0.05).结论 由于Flap-free Epi-LASIK特定的上皮去除方式导致了特定的上皮愈合方式,有利于减少术后刺激症状及角膜组织的正常修复.     

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.     

角膜疾病中细胞凋亡的作用机制

Dohlman CH等〔1〕三十年以前发现 ,前基质层的角膜细胞在上皮损伤后消失 ,当时被认为是由于机械损伤引起的组织暴露于周围环境及渗透压改变引起的。但很长时间因为检测仪器及检测方法的相对落后 ,对这个重要发现没有深入研究。 Campos M等〔2〕于九十年代初重新开始这一研究 ,证实上皮损伤后角膜细胞的消失是由细胞程序性死亡 (PCD)即凋亡引起的。凋亡是生物体在进化过程中去除不需要的细胞 ,而同时不释放降解酶引起周围组织细胞损伤的自身调节过程。凋亡与机体发育、体内环境稳定、感染、损伤和疾病的病理、生理变化都密切相关。角膜…     

单纯疱疹病毒性角膜炎中细胞凋亡的作用机制研究进展

单纯疱疹病毒性角膜炎（HSK）是一种严重的世界性致盲性角膜疾病,发病机制目前尚不明确。近年来的研究表明,该病的发病机制与细胞凋亡密切相关。病毒感染角膜后,可诱导角膜上皮细胞发生凋亡,随后抑制凋亡过程。同时亦可引起浅层基质发生细胞凋亡,以防止病毒从感染的上皮细胞传染至角膜深部。而当病变累及角膜深层时,常可发生角膜基质混浊、坏死、瘢痕形成、新生血管化以及角膜内皮皱褶等。就HSK发病过程中角膜上皮、基质、内皮发生细胞凋亡的机制进行综述。     

损伤相关性角膜基质混浊的研究进展

细胞凋亡、生长因子、炎细胞及细胞因子与损伤引起的基质混浊密切相关.在损伤愈合早期抑制角膜细胞的凋亡可减轻基质混浊,而在中后期适当地促进肌成纤维细胞及炎细胞的凋亡也有助于减轻基质混浊.目前研究发现,在众多的TGF-β信号转导通路中,Smad通路与角膜损伤后基质混浊有关.FAK、uPA/uPAR、EMMPRIN和CTGF作为TGF-β的下游活化产物均参与了角膜成纤维细胞向肌成纤维细胞的分化,其中FAK起负性调控作用,而后三者则起正性调控作用.炎症反应及其他参与损伤愈合的因素也是引起基质混浊的原因.阐明这些因素的作用机制,诱导其向着有益于损伤愈合的方向发展也是目前研究的主要内容.     

20%乙醇处理兔角膜后上皮增生和细胞凋亡的研究

目的探讨准分子激光角膜上皮瓣下磨镶术(LASEK)中采用20%乙醇浸润兔角膜40s后角膜上皮增生和角膜细胞凋亡情况与机械刮除角膜上皮后的异同。方法实验组42只新西兰大白兔,用直径为8mm的LASEK专用角膜上皮刀切割角膜上皮,20%的乙醇浸润单眼40s,机械刮除对侧眼中央8mm直径的角膜上皮,随机分7组,于术后0、4h,1、3、5、8、30d取材;6只兔眼为空白对照。角膜冰冻切片,行Ki67免疫组化检查和TUNEL检测,计数角膜中央前基质细胞。结果乙醇浸润后5d中央角膜上皮增生达峰值,术后1d周边角膜上皮增生达峰值;术后4h上皮刀口下方局限的角膜基质细胞TUNEL染色阳性,数量最多;各组角膜中央前基质细胞计数和空白对照比差异无统计学意义(P=0.68)。机械刮除角膜上皮后3d周边角膜上皮增生达峰值,其高于乙醇浸润后角膜上皮的增生峰值;术后4h可见大量中央前基质细胞TUNEL阳性;术后1d中央前基质细胞数量最少(P<0.05)。结论与机械刮除角膜上皮相比,20%乙醇浸润40s对角膜损伤轻,恢复快,乙醇浸润后的角膜上皮对基质细胞有保护作用。     

角膜创伤愈合中上皮细胞的作用初探

目的 探讨角膜创伤愈合中上皮细胞与角膜基质成纤维细胞的作用关系。方法 用形态学方法观察兔角膜损伤后上皮细胞与角膜基质成纤维细胞动态变化相关现象。结果 发现基质成纤维细胞的增生活跃程度与上皮基底细胞的细胞层数和细胞大小密切相关。结论角膜上皮细胞在角膜创伤个性中起主导作用。     

Wound healing in the cornea: a review of refractive surgery complications and new prospects for therapy

PURPOSE: The corneal wound healing response is of particular relevance for refractive surgical procedures since it is a major determinant of efficacy and safety. The purpose of this review is to provide an overview of the healing response in refractive surgery procedures. METHODS: Literature review. RESULTS: LASIK and PRK are the most common refractive procedures; however, alternative techniques, including LASEK, PRK with mitomycin C, and Epi-LASIK, have been developed in an attempt to overcome common complications. Clinical outcomes and a number of common complications are directly related to the healing process and the unpredictable nature of the associated corneal cellular response. These complications include overcorrection, undercorrection, regression, corneal stroma opacification, and many other side effects that have their roots in the biologic response to surgery. The corneal epithelium, stroma, nerves, inflammatory cells, and lacrimal glands are the main tissues and organs involved in the wound healing response to corneal surgical procedures. Complex cellular interactions mediated by cytokines and growth factors occur among the cells of the cornea, resulting in a highly variable biologic response. Among the best characterized processes are keratocyte apoptosis, keratocyte necrosis, keratocyte proliferation, migration of inflammatory cells, and myofibroblast generation. These cellular interactions are involved in extracellular matrix reorganization, stromal remodeling, wound contraction, and several other responses to surgical injury. CONCLUSIONS: A better understanding of the complete cascade of events involved in the corneal wound healing process and anomalies that lead to complications is critical to improve the efficacy and safety of refractive surgical procedures. Recent advances in understanding the biologic and molecular processes that contribute to the healing response bring hope that safe and effective pharmacologic modulators of the corneal wound healing response may soon be developed.     

Growth factors: importance in wound healing and maintenance of transparency of the cornea

The mechanism of corneal wound healing has not been clarified yet. However, evidence has accumulated that various kinds of growth factor such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF) play a key role in corneal wound healing. For example, these growth factors are expressed in the corneal epithelial cells, keratocytes and endothelial cells, and their receptors are expressed in the corneal cells. Furthermore, these growth factors promote the proliferation of corneal cells and induce the migration of corneal cells. In addition to the growth factors, inflammatory cytokines such as interleukin (IL)-1, IL-6 and TNF-alpha are involved in corneal wound healing. These cytokines are expressed in the normal and inflammatory cornea after infections, alkaliburn, etc. where they control the growth of corneal cells and induce the migration of corneal cells. Thus, a number of growth factors and cytokines function in the regulation of corneal cell proliferation and in the maintenance of corneal transparency.     

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.     

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 alkali wound healing in the monkey

Healing of a corneal alkali wound was followed in the monkey eye (Macaca fascicularis) for 4 months. A corneal alkali wound was inflicted in one eye in each of 30 monkeys under general anesthesia. A round filter paper, 5.5 mm in diameter, soaked in 1 N NaOH was put centrally on the cornea for 60 seconds. The alkali wounded all layers in the cornea underlying the filter paper, including the endothelium. Epithelial and endothelial healing was assessed morphometrically from photographs and micrographs, respectively. Stromal healing was documented using quantitative microradiography. During the observation period the wounds regained complete transparency. The epithelium showed few complications after primary healing during the first three days. The stroma regained normal thickness and dry mass content within one week. The endothelium resurfaced within one week and then remained intact. The role of the endothelial healing behaviour for the wound healing process is emphasized.     

Effect of ectopic epithelial tissue within the stroma on keratocyte apoptosis,mitosis, and myofibroblast transformation

The purpose of this study was to examine the effects of the epithelium on processes involved in stromal wound healing. Lamellar epithelial-stromal flaps were produced in rabbit corneas with a microkeratome. Peripheral corneal epithelial tissue, central corneal epithelial tissue, or no epithelial tissue (control) was introduced beneath the flap. Corneas were removed at time points from 4 hr to 1 month after surgery. Tissue sections were analyzed with immunocytochemistry for Keratin 3 (K3) to detect epithelial antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell proliferation, and immunocytochemistry for alpha-smooth muscle actin (SMA) to detect myofibroblasts. K3 was detected at the level of the interface from 4 hr to 1 month after surgery in corneas in which epithelial tissue was introduced, but not control corneas, with the exception of one that developed epithelial in growth. Keratocyte apoptosis was significantly higher at 4 hr after flap formation in both groups in which corneal epithelial tissue was introduced beneath the flap compared with controls. Keratocyte proliferation was significantly greater at 72 hr in corneas in which epithelial tissue was introduced beneath the flap compared to the controls. Corneas in which epithelial tissue was introduced into the interface, but not control corneas, had stromal cells expressing alpha-SMA in the stroma anterior and posterior to the interface at 1 week and 1 month after surgery. This was also noted in the control cornea in which there was epithelial ingrowth. Signals derived from the corneal epithelium promote keratocyte apoptosis. Keratocyte proliferation is higher in corneas that have lamellar surgery when epithelial tissue is introduced into the interface. Epithelium-derived signals also participate in the generation and/or maintenance of myofibroblasts in the corneal stroma.     

Evolution, techniques, clinical outcomes, and pathophysiology of LASEK: review of the literature

Laser subepithelial keratomileusis (LASEK) is a relatively new laser surgical procedure that combines certain elements of both laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) to improve the risk/benefit ratio. Diluted alcohol is used to loosen the epithelial adhesion to the corneal stroma. The loosened epithelium is moved aside from the treatment zone as a hinged sheet. Laser ablation of the subepithelial stroma is performed before the epithelial sheet is returned to its original position. We reviewed the literature regarding modifications of the technique, indications, outcomes, and complications, as well as wound healing after LASEK surgery. This literature review of 1,421 LASEK-treated eyes provided many findings: 1) The long-term stable results in the absence of serious complications, such as infections, recurrent erosions, scars, or late-onset corneal haze formation in patients re-examined up to 5 years after LASEK; 2) Epithelial closure with recovery of functional vision was completed at days 4 to 7 in most cases; 3) A tendency toward overcorrection with PRK nomograms; 4) We hypothesize that this tendency may be due to the decreased wound healing response, which may lead to myopic regression in PRK; and 5) Postoperative discomfort and prolonged visual recovery until the epithelium closes remain the biggest disadvantages of LASEK compared to LASIK. LASEK surgery is especially valuable in patients with thin corneas who would not qualify for LASIK surgery. However, a potential superiority of LASEK to LASIK in wavefront guided ablations still remains speculative.     

Growth factors in corneal wound healing following refractive surgery: A review

The first part of this review article aims to provide important basic definitions of growth factor terminology, and to put forward a model for understanding the role of growth factors in a wound healing context. In the second part of the paper, we review the literature on growth factors in the cornea, including that on changes associated with wound healing following refractive surgery in the epithelial, stromal, and endothelial layers. The role of growth factors in stromal removal, corneal neovascularization, corneal innervation and wound healing is considered. The importance of the epithelial-stromal interaction is discussed, including the role of growth factors in keratocyte apoptosis. In the final section, we review the current literature on endogenous and exogenous modulation of growth factors in corneal wound healing. This includes important in vitro work but aims to emphasize clinically relevant results. Photorefractive keratectomy (PRK) may have short-term complications such as pain and haze, whereas laser in situ keratomilieusis (LASIK) may have longer-term adverse effects on corneal biomechanics. The emerging technique of laser epithelial keratomilieusis (LASEK) provides an interesting alternative wound which may be less susceptible to the inherent complications of PRK and LASIK. At present, the phenomenon of iatrogenic keratectasia following LASIK is not fully understood, but these features of wound healing following PRK may be amenable to growth factor modulation.     

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.