血小板源性生长因子对角膜伤口愈合的调节

角膜损伤发生后，通过修复过程既可恢复角膜组织正常结构和功能，又可导致角膜瘢痕形成。由于角膜组织的特殊结构和功能引起了人们对促进角膜伤口愈合而又尽可能减少瘢痕形成研究的关注。角膜伤口的愈合涉及到细胞、细胞外基质及细胞间分子相互作用的复杂过程，且这一过程的关键在于调节。随着现代分子生物学技术的应用，已知细胞生长因子在调节伤口愈合过程中具有重要的作用。本文就血小板源性生长因子（platelet－derivedgrowthfactor，PDGF）对角膜伤口愈合的调节作一介绍。IPDGF的分类I‘DGF是细胞因子家族中发现较早的一类细胞因…     

角膜内皮创伤愈合研究的进展

角膜内皮创伤愈合早在19世纪就受到人们的重视,近年来由于各种新术学在眼科领域的应用,对角膜内皮愈合的形态、功能变化的动态过程、愈合机理、角膜移植后内皮细胞的变化均有了更深刻的认识。很多学者对愈合过程中内皮细胞的毗邻关系;细胞间连接、胞膜Na~+/K~+ ATP酶位点密度及它们与细胞形态、功能重建之间的关系;穿透性角膜移植后内皮细胞形态变化及其与细胞密度下降之间的关系;多肽生长因子对内皮细胞愈合的促进作用等进行了较系统的研究,对眼科临床医学的发展具有重要的指导意义。     

转化生长因子-β对角膜伤口愈合的调节

细胞生长因子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，且被人们所重视。本文叙述了转化生长因子－β（ＴＧＦ－β）及其受体的结构和作用，ＴＧＦ－β的激活和其在角膜细胞成分中的表达情况，同时讨论了ＴＧＦ－β在角膜伤口愈合过程中的作用。     

角膜内皮炎的内皮细胞测量分析

目的研究角膜内皮炎角膜内皮细胞的变化。方法用角膜内皮镜检查角膜内皮炎患眼的角膜内皮细胞,分析内皮细胞密度和形态的改变。结果角膜内皮镜检查26只患眼,显示内皮细胞密度降低,细胞异形性比率增高。其中10只眼在抗病毒治愈后随访期间无复发,角膜内皮细胞密度未见进一步下降。结论单纯疱疹病毒是角膜内皮炎的主要感染源,可引起角膜内皮细胞的破坏。     

PRK后兔角膜成纤维细胞分泌产生bFGF、TGF-β1及其对成纤维细胞增殖的影响

目的　探讨在准分子激光屈光手术后角膜伤口愈合过程中 ,是否有生长因子b FGF及 TGF- β1 参与 ,两种生长因子对成纤维细胞增殖的作用及其相应抗体对这一过程的影响。方法　对 PRK后兔角膜成纤维细胞原代培养 ,观察有否内源性生长因子 b FGF及 TGF-β1 的作用 ,内源性及外源性生长因子 b FGF及 TGF-β1 对细胞增殖的影响及相应抗体对抗原的中和作用。结果　在 PRK后兔角膜伤口愈合过程中成纤维细胞分泌产生b FGF及 TGF- β1 ,并刺激细胞的增殖 ,相应抗体可阻断这一生物效应。结论　 (1)准分子激光屈光手术后角膜愈合过程中成纤维细胞分泌产生生长因子 b FGF及 TGF- β1 ;(2 )两种生长因子对角膜成纤维细胞的增殖具有刺激作用 ;(3)相应抗体可阻断 b FGF及 TGF- β1 对细胞增殖的刺激作用。本研究结果提示抗 b FGF抗体及抗 TGF- β1 抗体作为准分子激光屈光手术后选择性用药的可能性 ,可防止或减少屈光回退和角膜上皮下混浊等两个主要并发症 ,提高手术效果     

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。     

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。     

青少年儿童角膜内皮细胞随年龄变化规律分析

目的:观察4~30岁青少年儿童角膜内皮细胞的密度和形态特点及内皮细胞随年龄变化的规律。方法:应用非接触型角膜内皮细胞显微镜进行观察。结果:随年龄增长,角膜内皮细胞密度降低,细胞面积增大,变异系数增大,六边形细胞百分率减少;年龄和各形态参数间存在高度相关性:细胞密度和六边形细胞百分率与年龄呈负相关,而平均细胞面积和变异系数与年龄呈正相关;内皮细胞密度与六边形细胞百分率呈负相关。结论:青少年儿童随着年龄的增长,角膜内皮细胞将发生一系列规律性变化。     

血小板活化因子致角膜上皮伤口迟愈合的实验研究

目的　利用兔去上皮角膜模型 ,研究血小板活化因子 (PAF)对角膜伤口愈合的作用及其分子生物学机制。方法　离体角膜上做正中直径 7mm圆形去上皮角膜伤口。去上皮角膜分为 3组 ,即对照、PAF及BN (PAF拮抗剂 )组 ,培养 4 8h后 ,行角膜上皮染色观察伤口愈合状况。分别对兔角膜上皮 (RCE)和角膜基质 (RCK)细胞进行体外传代培养 ,RCE和RCK细胞经PAF和 (或 )BN处理 ,培养 2 4h ,提纯RNA。应用RT PCR及核酸杂交技术分别检测肝细胞生长因子 (HGF)、角质形成生长因子 (KGF)及表皮生长因子 (EGF)基因在RCK和RCE细胞及HGF受体 (HGF R)基因在RCE细胞中的表达强度。分别应用CyQUANT荧光结合和Boyden小房技术检测PAF对RCE细胞黏附、增殖和迁徙的影响。结果　PAF (10 0nmol/L )明显抑制角膜上皮伤口愈合 ,4 8h对照、PAF和BN组角膜上皮未愈合面积经电脑图像分析分别为 :(6 0± 1.5 )U、(5 8 0± 7 0 )U和 (5 0± 1 0 )U。PAF明显增强RCE细胞黏附作用 ,对照、PAF和BN组每 96孔板贴附细胞数荧光光度平均值分别为 :36 96± 372、790 8± 6 71和 3487± 32 4。RT PCR结果显示 :PAF使HGFmRNA在RCK的表达强度降低 4 .1倍 ,同时明显减弱HGF R在RCE细胞中的表达 ,核酸杂交实验证实PCR结果。结论PAF明显增强RCE细胞的黏附作用 ,     

表皮生长因子对人角膜内皮细胞损伤修复的影响

目的　观察人表皮生长因子 (epidermal growth factor,EGF)对人角膜内皮细胞损伤修复的影响 ,检测人角膜内皮细胞表皮生长因子受体 (epidermal growth factor recep-tor,EGFR)的表达。方法　胎儿角膜内皮细胞体外培养传一代融合后 ,定量损伤细胞 ,倒置显微镜下观察不同浓度 EGF对角膜内皮细胞损伤修复的影响 ,并于损伤前及损伤后 1、3、7、14 d用间接免疫荧光的方法检测角膜内皮细胞 EGFR的表达。结果　 EGF促进人角膜内皮细胞损伤愈合 ,并显示剂量依赖性 ,EGF在 10 μg· L- 1时促进作用达高峰 ,浓度再增高促进作用下降。人角膜内皮细胞损伤前及损伤后均在细胞膜上表达 EGFR,表现为细胞膜上绿色荧光 ,在未损伤区的细胞及缺损区修复的细胞均见表达。结论　 EGF促进人角膜内皮细胞损伤修复 ,人角膜内皮细胞表达 EGFR     

Proliferative capacity of the corneal endothelium

Corneal endothelium is the single layer of cells forming a boundary between the corneal stroma and anterior chamber. The barrier and "pump" functions of the endothelium are responsible for maintaining corneal transparency by regulating stromal hydration. Morphological studies have demonstrated an age-related decrease in endothelial cell density and indicate that the endothelium in vivo either does not proliferate at all or proliferates at a rate that does not keep pace with the rate of cell loss. Lack of a robust proliferative response to cell loss makes the endothelium, at best, a fragile tissue. As a result of excessive cell loss due to accidental or surgical trauma, dystrophy, or disease, the endothelium may no longer effectively act as a barrier to fluid flow from the aqueous humor to the stroma. This loss of function can cause corneal edema, decreased corneal clarity, and loss of visual acuity, thus requiring corneal transplantation to restore normal vision. Studies from this and other laboratories indicate that corneal endothelium in vivo DOES possess proliferative capacity, but is arrested in G1-phase of the cell cycle. It appears that several intrinsic and extrinsic factors together contribute to maintain the endothelium in a non-replicative state. Ex vivo studies comparing cell cycle kinetics in wounded endothelium of young (< 30 years old) and older donors ( > 50 years old) provide evidence that cells from older donors can enter and complete the cell cycle; however, the length of G1-phase appears to be longer and the cells require stronger mitogenic stimulation than cells from younger donors. In vivo conditions per se also contribute to maintenance of a non-replicative monolayer. Endothelial cells are apparently unable to respond to autocrine or paracrine stimulation even though they express mRNA and protein for a number of growth factors and their receptors. Exogenous transforming growth factor-beta (TGF-beta) and TGF-beta in aqueous humor suppress S-phase entry in cultured endothelial cells, suggesting that this cytokine could inhibit proliferation in vivo. In addition, cell-cell contact appears to inhibit endothelial cell proliferation during corneal development and to help maintain the mature endothelial monolayer in a non-proliferative state, in part, via the activity of p27kip1, a known G1-phase inhibitor. The fact that human corneal endothelium retains proliferative capacity has led to recent efforts to induce division and increase the density of these important cells. For example, recent studies have demonstrated that adult human corneal endothelial cells can be induced to grow in culture and then transplanted to recipient corneas ex vivo. The laboratory work that has been conducted up to now opens an exciting new door to the future. The time is right to apply the knowledge that has been gained regarding corneal endothelial cell proliferative capacity and regulation of its cell cycle to develop new therapies to treat patients at risk for vision loss due to low endothelial cells counts.     

The coating of bovine and rabbit corneas denuded of their endothelium with bovine corneal endothelial cells.

A method for coating corneas denuded of their endothelium has been developed. The attachment and spreading of cultured bovine corneal endothelial cells seeded upon the Descemet's membrane of corneal buttons previously denuded of their endothelium by delicately sweeping the endothelial side with a cotton swab have been analyzed. Confluent cultures of bovine corneal endothelial cells were exposed to trypsin to disrupt the cell monolayer into single cells. Increasing concentrations of endothelial cells ranging from 2·5 × 10⁴ to 3 × 10⁵ cells were then seeded on the denuded Descemet's membrane of 11 mm bovine corneal buttons. When the corneal buttons were stained with alizarin red after an incubation period of 8 hr at 37°C, the best coating was observed with 10⁵ seeded cells. In this case, no areas of denudation could be seen and the cells were clearly apposed to one another, thereby reconstituting an endothelial cell monolayer. The cultured bovine corneal endothelial cells seeded on denuded Descemet's membranes plated extremely rapidly. By 15 min, 80% of Descemet's membrane was covered with a monolayer of endothelial cells and by 30 min all of Descemet's membrane was covered.The plating of bovine corneal endothelial cells on denuded Descemet's membrane was a direct function of the trypsin concentration to which they were first exposed. Cells first treated with 0·05% trypsin plated poorly 1 hr after being seeded on a denuded Descemet's membrane. Better plating efficiency was achieved with cells first exposed respectively to 0·025% and 0·01% trypsin. The best results were consistently obtained with cells first dissociated with 0·005% trypsin.Although serum is required in vitro for the attachment of normal cells to tissue culture dishes, it was not required for the attachment of corneal endothelial cells to the denuded Descemet's membrane. Cultured corneal endothelial cells plated equally well in the presence or absence of serum. Similar results were obtained when the cells were suspended in aqueous humor instead of in tissue culture medium.When denuded rabbit corneas were used as a substrate instead of bovine corneas, all the parameters studied for the attachment and spreading of bovine corneal endothelial cells seeded on bovine corneas (cell density, time, and medium) lead to similar conclusions with respect to the interactions between corneal endothelial cells and rabbit Descemet's membrane.     

生长因子诱导的血管内皮细胞增殖作用的研究

目的 研究生长因子对血管内皮细胞增殖活动的诱导和刺激作用。以进一步探讨生长因子在视网新生血管疾病中所起的作用。方法 采用血管内皮生长因子（VEGF）和碱性成纤维生长因子（bFGF）诱导离体培养的血管内皮细胞的增殖。研究VEGF及bFGF对血管内皮细胞DNA合成、细胞生长和细胞周期的调控，以及两种因子的协同效应。结果 VEGF及bFGF均可显著刺激血管内皮细胞的增生，其刺激效应与生长因子浓度呈剂量依赖关系。共同作用时，具有协同效应。两种因子均可显著增加^3H-TdR的掺入量，其刺激作用与浓度呈剂量依赖关系，共同作用时亦具有协同效应。VEGF和bFGF均可显著地产加S期细胞的比例。结论 VEGF及bFGF均可显著地刺激内皮细胞的增殖，促进细胞DNA的合成。两种因子联合应用时，具协同效应。提示：两种生长因子在视网膜新生血管的发生和发展中可能具有一定作用，且存在因子间的协同效应。     

Factors affecting bovine corneal endothelial cell density in vitro.

AIMS: To examine factors influencing the density and contact inhibition of bovine corneal endothelial cells cultured in vitro. METHODS: Cell counts were performed on bovine corneal endothelial cells cultured for various times in the presence of 10% fetal calf serum, with or without varying concentrations of growth factors, 5% dextran T-500, or 2% chondroitin sulphate, at 32 degrees C or 37 degrees C, and after treatment with beta galactosidase. RESULTS: Both basic fibroblast growth factor (FGFb) and retinal crude extract (RCE), but neither epidermal growth factor (EGF) nor acidic fibroblast growth factor (FGFa), increased endothelial cell density in vitro (p < 0.05). Continuous exposure to RCE resulted in a higher cell density than did a 24 hour pulse (p < 0.01), and higher cell densities were achieved at 37 degrees C than at 32 degrees C (p < 0.0001). In the absence of RCE, dextran T-500 increased cell density modestly (p < 0.05); in the presence of RCE, the addition of dextran T-500 had no effect on final cell density, whereas chondroitin sulphate significantly decreased final cell density (p < 0.01). In the absence of exogenous growth factors, beta galactosidase treatment resulted in a 50% increase in final cell density compared with controls (p < 0.0001). CONCLUSIONS: Bovine corneal endothelial cell growth can be augmented under conditions different from those used in corneal preservation systems. The final cell density in a confluent monolayer can be increased by treatment with beta galactosidase, suggesting that corneal endothelial cells may be contact inhibited through a beta galactosidase sensitive receptor system.     

The effects of epidermal and fibroblast growth factors on the repair of corneal endothelial wounds in bovine corneas maintained in organ culture.

The effects of FGF and EGF on the repair process of the wounded endothelium of bovine corneas maintained in organ culture have been analyzed. Both EGF and FGF greatly accelerate the repair process of the corneal endothelium in vitro. Similar results were obtained when complete denudation of the endothelium was performed and cultured corneal endothelial cells were seeded at a final density of 3×10³/cm² on Descemet's membrane. Within 5 days a new endothelium with a morphology similar to that of intact corneal endothelium maintained in organ culture for the same period time was achieved. These results demonstrate, therefore, that either EGF or FGF can participate in the repair process of the corneal endothelium of corneas maintained in organ culture.     

Endothelial cell transplantation and growth behavior of the human corneal endothelium

Background: The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells, transplantation of these cells my be an alternative therapeutic option. Materials and methods: In this review methods for the in vitro cultivation of human corneal endothelial cells and their transplantation on the Descemet membrane of donor corneas are described. Results: In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors and substances. Dependent on the culture conditions, as well as independent of them, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behavior. Thus, molecular biological examinations revealed a different expression pattern of growth factor receptors in fibroblast-like endothelial cells (dedifferentiated) compared to typical endothelial cells (differentiated). Moreover, the proliferative capacity of the cells differed, dependent on their corneal location. Cells isolated from the peripheral part of donor corneas have a higher proliferative capacity than cells obtained from the central part. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation on donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation of the cells. DNA synthesis was predominantly detectable in cells of the corneal periphery. Conclusions: Our findings are the basis of the following hypothesis: the periphery of the cornea represents a regenerative zone of the corneal endothelium. The fact that early after transplantation corneal endothelial cells form a monolayer on the natural extracellular matrix (ECM), which shows contact inhibition, suggests that inhibitory factors are released by the Descemet membrane that influence the proliferation of the cells. Further studies on the regulation of the proliferation and differentiation of human corneal endothelial cells in vitro and after transplantation might offer the possibility to establish a selective procedure for the treatment of corneal endothelial cell loss in the near future.      

Regeneration of cat corneal endothelium induced in vivo by fibroblast growth factor

The corneal endothelium of man, primates and carnivores has limited regenerative ability. Fibroblast growth factor (FGF) stimulates the proliferation of various mesoderm-derived cells, including corneal endothelial cells, in vitro. In the present work the effect of FGF on the regeneration of injured cat endothelium in vivo was studied. Scraping of cat corneal endothelium off Descemet's membrane was carried out by a specially designed instrument. FGF from crude preparations (25 micrograms in 50 microliter saline) or affinity purified FGF (0.5 micrograms in 25 microliter saline) were injected immediately after the scraping of the endothelium, and in both cases significant stimulation of endothelium regeneration was observed. During the first 2 weeks after scraping, the endothelial cell density (ECD) in FGF-treated eyes was higher by a factor of 1.7-2.3 than that in the control eyes. This marked difference decreased with time, but even after 12 weeks the ECD in FGF-treated eyes was higher by a factor of 1.15-1.30 than in the control eyes. Furthermore, FGF improved the polygonal shape of the cells and decreased the corneal thickness. These results clearly demonstrate the efficacy of FGF in inducing the proliferation of cat corneal endothelial cells in vivo and indicate its potential application in clinical practice.     

The study of human PDGF-B gene transferred to cat corneal endothelial cells

AIM: To demonstrate that human platelet-derived growth factor-B (PDGF-B) cDNA could be expressed in primary cultured cat corneal endothelia cells by using gene transfer techniques; to explore a useful tool for the further studies of the molecular mechanisms of corneal endothelium failure and provide a potential effective genetic therapy for the blind patients. METHODS: Human PDGF-B cDNA was isolated from human placent by RT-PCR and inserted into pcDNA4 vector to construct recombinant eukaryotic expression plasmid pcDNA4-PDGF-B. The full length was confirmed by the DNA sequencing analysis. By tearing endothelium technique we obtained pure single layer of cat corneal endothelial cells. The pcDNA4-PDGF-B eukaryotic expression vector was transferred into cat corneal endothelial cells by EffecteneTM lipofectine. The transfection efficiency of EffecteneTM lipofectine in pcDNA4-B was detected with pcDNA4-GFP. 5 days later, RT-PCR was used to check the PDGF-B expression. Cell viability was tested by modified tertrozalium salt (MTT) method. Cell morphology was observed under inverted phase contrast microscope. RESULTS: The human PDGF-B cDNA was isolated successfully from healthy parturien placent tissue and the sequence was confirmed by computer automatic sequence and PCR analysis. Pure single layer cat corneal endothelial cells were successfully cultured by tearing endothelium technique. EffecteneTM lipofectine transfection technique could be effectively used to transfer pcDNA4-PDGF-B into cat corneal endothelial cells in vitro, the transfection efficiency was 30%. RT-PCR result showed that human PDGF-B gene was highly expressed in transfected cat corneal endothelial cells. The expressed PDGF-BB protein promoted the viability of cat corneal endothelial cells. CONCLUSION: Human platelet-derived growth factor-B (PDGF-B) cDNA could be highly expressed in cultured cat corneal endothelial cells by gene transfection techniques. Expressed PDGF-BB protein significantly promoted the viability of cat corneal endothelial cells, thus provided a potential effective method for corneal endothelium blindness genetic therapy.     

Retinoid-induced potentiation of epidermal growth factor mitogenic effect on corneal endothelial cells

Epidermal growth factor (EGF) is mitogenic for bovine corneal endothelial cells in culture. Pretreatment with either retinoic acid or the synthetic analog CBS-211 A ((E)-4-[2-(2-isopropyl-5-thienyl)propenyl]benzoic acid) at 10(-8)-10(-6) M enhanced the EGF effect. This potentiating effect of retinoids contrasts with their intrinsic activity, which results in cell growth inhibition in the absence of growth factor. The present data emphasize the previously reported beneficial effect of retinoic acid on corneal endothelium wound healing. This effect could be related to the potentiation of endogenous growth factors and further underlines the importance of retinoids in the medical treatment of corneal endothelial lesions.     

Ex vivo transfer of Smad7 decreases damage to the corneal endothelium after penetrating keratoplasty

PURPOSE: To determine whether transient gene transfer and expression of the intracellular antagonist of transforming growth factor beta (TGF-beta), Smad7, to corneal endothelial cells decreases corneal endothelial cell damage after penetrating keratoplasty in a rabbit model. METHODS: Rabbit corneas were transfected ex vivo with replication-deficient adenoviruses encoding Flagtagged Smad7, Flag-tagged Smad3, or LacZ (termed AdCMV-Smad7, AdCMV-Smad3, AdCMV-LacZ) and then transplanted to normal rabbits. Expression of the exogenous Smads and phosphorylation of endogenous Smad2 in the transplanted corneal endothelium were examined by immunoblotting and immunohistochemistry with anti-Flag or anti-phosphorylated Smad2 antibodies. Cellular density and morphological changes in the corneal endothelium of the transplanted cornea were evaluated by scanning electron microscopy after transplantation of the Smad-transfected corneas. RESULTS: Transplanted AdCMV-Smad7-transfected corneas significantly inhibited the decrease in cellular density and accelerated wound healing at the host-graft junction when compared with transplanted AdCMV-LacZ-transfected corneas. Transplanted AdCMV-Smad3-transfected corneas showed decreased cellular density and delayed wound healing at the host-graft junction. CONCLUSIONS: Ex vivo gene transfer of Smad7 to corneal endothelial cells inhibits the decrease in cellular density and accelerates wound healing after penetrating keratoplasty in rabbits. Thus, modulation of Smad7 expression in corneal endothelial cells may decrease corneal endothelial cell damage after penetrating keratoplasty.