Arachidonic acid metabolism by cultured bovine corneal endothelial cells

Pathways of arachidonic acid metabolism were identified in freshly prepared and in cultured bovine corneal endothelial cells. The principal pathway of arachidonic acid metabolism in the bovine corneal endothelial cells appears to be the cyclooxygenase pathway with the resultant synthesis of PGI2, PGF2 alpha and PGE2. At least two of these products, PGI2 and PGF2 alpha, are formed by the enzymatic conversion of the substrate, PGH2. Measurements of endogenous prostaglandin production by radioimmunoassay demonstrated that PGE2 was the major arachidonic acid metabolite released, with smaller amounts of PGF2 alpha and the stable hydrolysis product of PGI2, 6-keto PGF1 alpha. The release of all three prostanoids was significantly increased by the addition of the calcium ionophore (A23187), human thrombin, bradykinin and histamine. Basal and stimulated release of prostaglandins by the corneal endothelium may contribute to the regulation of intraocular pressure and also in the modulation of the corneal response to injury.     

Maintenance of corneal endothelial cell shape by prostaglandin E2: effects of EGF and indomethacin

Confluent, cultured, rabbit corneal endothelial cells maintain a polygonal shape which is characteristic of these cells in vivo. When cultured in the presence of EGF (10 ng/ml) and/or indomethacin (1.0 microM), the endothelial cells have markedly different shapes at confluency. By morphometry, untreated cells are polygonal and have a maximum axis of 33 mu; EGF treatment causes a spindle-shaped elongation to 48 mu and indomethacin treatment causes a stellate-shaped elongation to 48 mu. There is a slight increase in cell density. When cells are cultured in the presence of both drugs, elongation is more pronounced to a fibroblastic appearing cell population, with maximum axes of 60 mu and more, but no additive increase in cell density. Continuity of cell borders is often lost. Corneal endothelial cells cultured in the presence of EGF, indomethacin, and PGE2 (0.5 microgram/ml) maintain their polygonal shape; PGF2 alpha is not effective at reversing the drugs' effects. Untreated and EGF-treated cells synthesize and release substantial quantities of PGE2 (2-4 ng/10(4) cells). Indomethacin completely inhibits PGE2 synthesis. It is concluded that PGE2 maintains the polygonal cell shape of the corneal endothelium in vitro and, perhaps, in vivo. The elongated forms of the cell may be related to migration and important in wound closure.     

Effect of copper ion on collagenase release. Its implication in corneal vascularization.

The involvement of leukocytes in corneal neovascularization has been known for a long time. Recent observations suggest that collagenase from leukocytes may be a common mediator for prostaglandin E1 (PGE1)- and copper-induced corneal neovascularization. This study was designed to investigate the effect of copper ion on collagenase activity from leukocytes and other sources and leukocyte infiltration in the corneal angiogenic process induced by PGE1. These results demonstrated that collagenase production from leukocytes was stimulated in a dose-dependent manner by copper ion but not by PGE1. Copper chloride 0.2 mM produced the highest stimulation. Copper ion had no effect on collagenase release from corneal fibroblasts and capillary endothelium. There were more polymorphonuclear leukocytes (PMN) in the prostaglandin E1 treated corneas than in the control. The time-course study showed that the appearance of PMN reached a peak on day 2 and new vessel growth could not be identified until day 4. These results supported an earlier suggestion that leukocytes play a role in corneal neovascularization and further suggested that copper in corneal neovascularization can stimulate the release of collagenase from leukocytes.     

Transforming growth factor-beta2 inhibition of corneal endothelial proliferation mediated by prostaglandin

PURPOSE: To determine the influence of Prostaglandin (PG) E2 on transforming growth factor (TGF)-beta2-mediated inhibitory effects on the proliferation of corneal endothelial cells (CE). METHODS: The PGE2 and cell proliferation assays were performed using cultured rabbit corneal endothelium. A PGE2-specific enzyme immunoassay was used to check PGE2 synthesis in supernatants of cells cultured with and without added TGF-beta2 and/or indomethacin. To evaluate the inhibitory effects of PGE2 and TGF-beta2 on CE proliferation, the number of cells grown with exogenous PGE2, or TGF-beta2 with or without indomethacin pretreatment was determined. RESULTS: TGF-beta2, 0.5 to 50 ng/ml, increased the PGE2 secretion of CE dose-dependently in a time-dependent manner. Indomethacin (> or =0.1 microg/ml) inhibited this PGE2 secretion to a low level (around 5-10 ng/ml) in the presence or absence of exogenous TGF-beta2. Both exogenous TGF-beta2 and PGE2 inhibited CE proliferation dose-dependently over a wide range of concentrations. Indomethacin reversed the inhibitory effects of TGF-beta2 but not those of exogenous PGE2. In the medium supplemented with indomethacin, even in the presence of 50 ng/ml of TGF-beta2, CE growth did not differ from control cultures. CONCLUSIONS: TGF-beta2 stimulates PGE2 synthesis in CE and inhibits CE proliferation in a dose-dependent manner. Indomethacin extinguishes the inhibitory effects of TGF-beta2 on CE proliferation but not the effect of exogenous PGE2. These data suggest that the antiproliferative effects of TGF-beta2 on CE may be possibly due to TGF-beta2-induced synthesis of PG, most likely PGE2. SUMMARY: Inhibition of endogenous prostaglandins synthesis by indomethacin extinguished the inhibitory effects of Transforming Growth Factor-beta2 on corneal endothelium proliferation but not exogenous prostaglandin E2. It suggesting that TGF-beta2-induced autocrine synthesis of PGs, most likely PGE2, may be responsible for the anti-proliferative effects of TGF-beta2 on corneal endothelium.     

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。     

Effect of latanoprost on cultured porcine corneal stromal cells

PURPOSE: Latanoprost reduces intraocular pressure mainly by enhancing uveoscleral outflow that may be involved in the decreased of extracellular matrixes such as collagens. However, the effect of latanoprost on corneal stromal cells is not well understood. In the current study, we investigated the changes of cultured porcine corneal stromal cells upon exposure to latanoprost. METHODS: Porcine corneal stromal cells were acquired from primary culture and maintained in fetal bovine serum-containing medium. Cells were estimated on 3H-thymidine, 3H-leucine, 3H-uridine, 3H-proline uptakes and migration. Dead and living cells were estimated with MTT assay. The changes of type 1 collagen and fibronectin proteins were detected by means of immunofluorescent staining and Western blot assay. Intracellular free Ca2+ ([Ca2+]i) mobility was studied by spectrofluorophotometer after loading with fura-2-AM. RESULTS: Latanoprost has remarkable effects inhibiting cultured corneal stromal cells on 3H-thymidine, 3H-leucine, 3H-uridine, 3H-proline uptakes and cellular migration. The inhibitory effects are in a dose-dependent manner at concentrations ranging from 10(- 5), 10(- 6), 10(- 7) to 10(- 8) M. The 50% inhibitory dosages (ID50) for latanoprost to corneal stromal cells, as measured by 3H-thymidine uptake, 3H-uridine uptake, 3H-leucine uptake, 3H-proline uptakes and cellular migration were 5.01 x 10(- 6) M, 2.81 x 10(- 6) M, 2.09 x 10(- 6) M, 3.89 x 10(- 7) M and 2.2 x 10(- 6) M, respectively. In the presence of latanoprost, the cellular MTT values were also decreased significantly. Immunofluorescent staining displayed that latanoprost changed type 1 collagen distribution in cultured corneal stromal cells. Western blot assay revealed that latanoprost caused cells to decrease in fibronectin protein. In Ca2+-containing buffer, latanoprost induced a significant rise in [Ca2+]i at 10(- 5) and 10(- 6) M. CONCLUSIONS: These results indicate that latanoprost may induce the morphological and biochemical changes in cultured corneal stromal cells. Long-term use of latanoprost needs to be carefully monitored for change in corneal stroma.     

Effect of neuraminidase on Fc and C3b receptors on rabbit corneal cells infected with herpes simplex virus

The effect of neuraminidase on Fc receptors (FcR) and C3b receptors (C3bR) was studied in epithelial, stromal and endothelial cells of the rabbit cornea infected with type 1 (HSV-1) and type 2 herpes simplex virus (HSV-2) in vitro. FcR were induced on epithelial, stromal and endothelial cells of the rabbit cornea by both HSV-1 and HSV-2, but their activities were not enhanced by neuraminidase. On the other hand, the treatment of HSV-infected corneal cells with neuraminidase resulted in the enhancement of C3bR activities on epithelial, stromal and endothelial cells infected with HSV-1, and the enhancing effect of neuraminidase was more pronounced on corneal endothelial cells. A similar neuraminidase treatment had no significant effect on C3bR activities on the corneal cells infected with HSV-2.     

Tierexperimentelle untersuchungen zur rolle von entzündungsmediatoren bei der hornhautneovaskularisation

Experimental investigations concerning the role of inflammatory compounds in corneal neovascularization.Natural and synthetic inflammatory compounds were implanted in the corneas of rabbits to clarify the question whether corneal neovascularization is induced by stromal edema alone, or by neovascular mediators. It could be demonstrated that prostaglandin E1 and E2 have an angiogenetic capacity, whereas their precursor (arachidonic acid) as well as PGA1, A2, B2, I2 and Thromboxan A2 were inactive in this regard.Histology showed that corneal neovascularization is always accompanied by the invasion of polymorphonuclear leukocytes. Corneal edema in the beginning of vascularization can be explained by the activities of PGE (vasodilation, increase of vascular permeability, liberation of histamine). The implantation of lipoxygenase-dependent arachidonic acid compounds (5-HETE, Leukotriene B4) demonstrated that these mediators share in the process of neovascularization by inducing the chemotaxis. The above mentioned activities of prostaglandins and leukotrienes could also be demonstrated following penetrating keratoplasty and alkali burns of the anterior segment inducing extensive corneal neovascularization. An analysis of the prostaglandin- and leukotriendependent mechanisms could be achieved by selective PG- and LT- inhibitors. Radioimmunoassays showed a definite correlation between the concentrations of PGE and the amount of neovascularization following alkali burns. The results of our research lead to the following scheme of pathophysiology of corneal neovascularization: hypoxic, chemical, thermic and mechanical alterations of the cornea induce an activation of corneal cytomembranes, thus initiating (1) the cyclooxygenase-dependent synthesis of prostaglandins with consecutive vasodilation and increase of vascular permeability as well as histamine liberation resulting in corneal edema; on the other hand, prostaglandins proved to have a minimal chemotactic activity; (2) the lipoxygenase-dependent synthesis of leukotrienes inducing chemotaxis and diapedesis of polymorphonuclear leukocytes into the corneal stroma. These inflammatory cells are then the main source of newly synthesized leukotrienes maintaining the chemotaxis, and prostaglandins with angiogenetic activity. Cyclooxygenase- and lipoxygenase- inhibitors can inhibit these activities at two different levels, leading to an approach of successful therapy of corneal dieases inducing neovascularization.

     

Latanoprost does not affect immune privilege of corneal allografts

The present study determined whether topical latanoprost, a prostaglandin (PG) F(2alpha) analog, influences the induction of anterior chamber-associated immune deviation (ACAID), corneal neovascularization (NV) or survival of corneal allografts in mice. BALB/c mice received topical latanoprost or PGE(2) once or multiple times daily starting 4 weeks prior to or the day of anterior chamber injection of C57BL/6 splenocytes. Induction of allo-specific ACAID was assessed by ear challenge with C57BL/6 splenocytes 1 week after subcutaneous immunization. In a separate experiment, orthotopic corneal transplantation was performed using C57BL/6 mice as donors and BALB/c mice as recipients. Recipients were randomized in a masked fashion to receive topical latanoprost or PGE(2). Graft fate was assessed clinically under surgical microscopy. Presence of MHC class II(+) CD11c(+) or CD11b(+) cells in normal BALB/c mouse eyes following latanoprost or PGE(2) administration was assessed immunohistochemically. Control mice received topical 20% dimethyl sulfoxide or no treatment. Allo-specific ACAID was induced after 2 or 6 weeks of once daily treatment with latanoprost, and was induced even after 6 weeks of multiple treatments with latanoprost. Conversely, mice receiving PGE(2) failed to develop ACAID. Opacity and corneal NV scores for allografts treated with latanoprost were statistically indistinguishable from those for control allografts (p>0.05), whereas all allografts treated with PGE(2) were rejected. Opacity and NV scores were significantly higher in these allografts than in controls (p<0.05). A number of MHC class II(+) CD11c(+) cells were present in the central cornea after PGE(2) treatment. Topical application of latanoprost does not influence induction of ACAID or graft outcomes including opacity and NV, whereas PGE(2) does. Immune privilege of corneal allograft is maintained after topical latanoprost application in mice.     

Antifungal chemotherapy for fungal keratitis guided by in vivo confocal microscopy

Background To evaluate antifungal chemotherapy in patients with fungal keratitis guided by in vivo confocal microscopy. Methods A total of 121 patients (121 eyes) with fungal keratitis were enrolled in this study. Confocal microscopy was performed in real time after topical and/or oral antifungal chemotherapy. Hyphal density and morphology, composition of inflammatory cells, and appearance of corneal stromal cells at the central and peripheral corneal lesions were recorded. Antifungal therapy discontinued at 1 week after hyphae and inflammatory cells could not be detected, and affected corneal stromal cells became visible. Results Successful outcomes were achieved in 110 patients (90.9%). By confocal microscopy, we observed the gradual decrease of hyphae-positive sites and hyphal density during the chemotherapy. The inflammatory cells reduced in number and heterogeneity, while corneal stromal cells recovered. The antifungal drugs were tapered according to the changes in hyphae, inflammatory cells, and corneal stromal cells. There was no fungal recurrence during the 2-month follow-up period. The other 11 patients (9.1%) had deteriorated infection within 1 week of antifungal therapy, and therefore were subjected to corneal transplantation. Conclusions In vivo confocal microscopy appears to be an effective approach to guide antifungal chemotherapy. It allows comprehensive evaluation of hyphae, inflammatory cells, and corneal stromal cells in real time, and provides valuable and objective information required in selecting and adjusting therapeutic regimens for the treatment of fungal keratitis.     

Contribution of virus and immune factors to herpes simplex virus type I-induced corneal pathology

In vivo T-lymphocyte subpopulation depletion techniques were used to identify the roles of L3T4+ (CD4) and Lyt-2+ (CD8) T-lymphocytes in the pathogenesis of corneal stromal disease induced by two different strains of Herpes simplex virus type 1 (HSV-1). Histologic examination of infected corneas revealed significant differences in the composition of the inflammatory corneal infiltrates induced by the RE and KOS strains of HSV-1. The RE strain induced a predominantly polymorphonuclear leukocyte (PMN) infiltrate, which began approximately 1 week after infection and progressed through day 21. Depletion of CD4 cells before corneal infection with RE HSV-1 greatly reduced the incidence and severity of corneal disease; depletion of CD8 cells had no effect. The strain KOS HSV-1 induced an early PMN infiltrate that became predominantly mononuclear by day 21. Depletion of CD4 cells did not change the incidence or severity of KOS HSV-1-induced corneal stromal disease. The corneal lesions of these mice contained numerous CD8 cells. Depletion of CD8 cells before KOS HSV-1 infection of the cornea moderately reduced the incidence of stromal disease. However, in CD8-depleted mice with the disease, PMNs were the most prevalent infiltrating cells, and the disease appeared identical to that seen in RE HSV-1 infected corneas. Simultaneous depletion of CD4 and CD8 cells before KOS HSV-1 infection eliminated stromal disease. However, when T-cell depletion was discontinued in these mice, stromal disease developed in concert with the appearance of T-cells in the lymphoid organs and corneas. Thus, T-lymphocytes are a necessary component of HSV-1 corneal stromal disease. These results further suggest that RE HSV-1 preferentially activates CD4 cells in the cornea, and KOS HSV-1 preferentially activates CD8 cells in the cornea.     

RNA干扰对兔角膜基质细胞环氧化酶2与基质金属蛋白酶2表达的抑制作用研究

目的 探讨针对环氧化酶2(COX-2)的RNA干扰(RNAi)对兔角膜基质细胞中COX-2与基质金属蛋白酶2(MMP-2)表达的影响.方法 实验研究.体外培养兔角膜基质细胞,测定阳离子脂质体介导的小干扰RNA(siRNA)在兔角膜基质细胞中的转染率,设计并合成3条针对兔COX-2基因的短发卡RNA(shRNA)1、2、3和1条阴性对照shRNA,利用阳离子脂质体介导转染正常与IL-lα刺激后的兔角膜基质细胞,在IL-lα刺激后6、12、24、48、72 h收集细胞,实时荧光定量聚合酶链反应检测兔角膜基质细胞中COX-2和MMP-2基因的表达变化,并与对照组进行比较.同一时间点各组间比较采用单因素方差分析,处理组两两比较采用q检验.结果 阳离子脂质体能够有效介导siRNA转染体外培养的兔角膜基质细胞,转染率可达70%～80%.IL-lα刺激后兔角膜基质细胞中COX-2与MMP-2 mRNA的表达较空白对照组显著升高;在不同时间点,生物合成的3条针对COX-2的靶向shRNA中shRNA-2能显著抑制IL-α刺激后的兔角膜基质细胞中COX-2、MMP-2 mRNA表达,抑制率最高可分别达83.04%、73.69%,与单纯IL-lα刺激组相比差异均有统计学意义(q=24.03,P=0.00;q=14.76,P=0.00);而shRNA-1、shRNA-3、阴性对照shRNA转染组与单纯IL-lα刺激组相比,COX-2 mRNA的表达差异无统计学意义(F=0.02,P=0.99),MMP-2 mRNA的表达差异也无统计学意义(F=0.02,P=0.98).结论 针对COX-2的RNAi能够有效抑制IL-lα诱导后的兔角膜基质细胞中COX-2与MMP-2的表达.     

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。     

Expression of NADPH oxidase in rabbit corneal epithelial and stromal cells in culture

PURPOSE: Reactive oxygen- and nitrogen-containing molecules produced in high concentrations are mediators of tissue damage caused by inflammation. The free radical molecules superoxide (O2-*) and nitric oxide (NO*), when produced at low concentrations, may function as second messengers or regulators of signal transduction. The purpose of these studies was to determine whether corneal epithelial and stromal cells are capable of producing O2-* via an NADPH oxidase complex. METHODS: Rabbit corneal epithelial and stromal cells, grown as primary cultures and low-passage isolates, were used as the sources of RNA for RT-PCR with primers specific for mRNAs encoding the proteins that comprise an NADPH oxidase complex. The RT-PCR products were sequenced to confirm their identities. The production of proteins composing the oxidase complex was confirmed, and the proteins were identified by Western blot analysis. The production of superoxide in cell-free preparations was assessed by measurement of NADPH-dependent superoxide dismutase (SOD)-inhibitable cytochrome c reduction and by electron paramagnetic resonance (EPR) with a superoxide specific spin trap. RESULTS: Cell-free extracts of corneal epithelial and stromal cells produced superoxide in an NADPH-dependent manner, and this production was inhibited by SOD. EPR confirmed the identity of the reaction product as superoxide anion. Both rabbit corneal epithelial and stromal cells constitutively produced mRNAs encoding five proteins known to comprise a classic neutrophil-like NADPH oxidase complex. Production of NOX4, p22phox, p47phox, p67phox, and p40phox was confirmed by Western blot. Both epithelial and stromal cells expressed isoforms of Rac, a putative regulator of the activity of the complex. CONCLUSIONS: A constitutively expressed NADPH oxidase complex that includes NOX4 is a source of O2-* produced by rabbit corneal epithelial and stromal cells. Superoxide produced by the oxidation of NADPH via the NADPH oxidase complex is a potential contributor to signal transduction pathways as well as a potential participant in processes that occur during inflammation.     

An intermediate filament-associated developmentally regulated protein in corneal fibroblasts

A developmentally regulated, cytoskeletal-associated protein was identified and partially characterized using a monoclonal antibody developed for this study. Based on the distinctive fibrous pattern of distribution of this antigen in the cytoskeletons of cultured corneal fibroblasts, and a characteristic reorganization of these fibers into perinuclear whorls in response to colchicine treatment, this protein was found to be associated with the intermediate filaments (vimentin filaments). Chronological distribution of this intermediate filament-associated protein (IFAP) and vimentin during fetal development of the cornea in rabbit was analysed immunohistochemically. During an early stage of corneal development (day 13 of gestation), both this IFAP and vimentin were present in the stromal cells in the presumptive corneal region. The IFAP was also present in the surface epithelium. At day 17 and 21, the corneal endothelial layer was developed and contained both the IFAP and vimentin. However, in the corneal stromal cells, the concentration of IFAP (based on the densities of the immunostaining reaction) progressively decreased during the later stages of fetal development (day 24 to day 28), while relative concentrations of vimentin were not reduced significantly. In the corneal stromal cells in the adult rabbit, the IFAP was not detectable while vimentin was still present. For further characterization of this protein, extracts of cultured corneal fibroblasts and fetal corneas were analysed by SDS-PAGE followed by an immunotransblot technique. These analyses indicated that the IFAP was a polypeptide with a Mr of 130k. Therefore, this protein was not vimentin (Mr 55-58k). The presence of this IFAP in the fetal corneas and stromal cells and its absence in the quiescent stromal cells in the adult cornea indicated that this unique IFAP is developmentally regulated in corneal stromal cells, and its association with vimentin filaments may be important during the active state of corneal stromal cells.     

雌激素对人角膜基质细胞MMP-2、TIMP-2及TGF-β1表达的影响

目的:在体外实验中探讨雌激素对人角膜基质细胞中基质金属蛋白酶MMP-2及其抑制剂TIMP-2和转化生长因子TGF-β1蛋白表达的影响.方法:用1.5ng/mL IL-1β模拟角膜基质细胞炎性环境,不同浓度雌激素(0,10-10,10-8,10-6,10-4mol/L的17-β雌二醇)体外作用于人角膜基质细胞,四甲基偶氮唑(MTT)比色法鉴定细胞活性.酶联免疫吸附测定(ELISA)法检测各组细胞上清中MMP-2、TIMP-2及TGF-β1蛋白的表达量.结果:不同浓度雌激素(E2)对角膜基质细胞的活性没有影响.E2处理组的MMP-2、TIMP-2及TGF-β1表达量与对照组比较,差异均有统计学意义(P<0.05).E2处理组与对照组相比,雌激素处理使MMP-2及TGF-β1的表达明显减少,而TIMP-2的表达则显著增多.结论:雌激素能一定程度抑制MMP-2及TGF-β1的表达,同时促进TIMP-2的表达,这可能对正常人角膜起保护作用.     

角膜基质修复的机制和调控因素研究进展

角膜基质是维持角膜透明度的重要结构。外伤、感染、手术等可造成角膜基质损伤,引起修复的过程包括基质细胞表型改变、细胞外基质重塑、免疫细胞迁移。当基质严重受损,肌成纤维细胞增多和细胞外基质沉积发生基质纤维化反应,形成角膜瘢痕,是全球致盲的主要原因。目前治疗方式主要是角膜移植手术,因角膜供体资源短缺、手术技巧要求和术后移植排斥风险等治疗效果不佳。近年来,各种分子、细胞和组织对角膜基质损伤修复的调控机制取得一定研究进展。本文就角膜基质损伤修复的机制和角膜损伤原因、角膜结构、分子因素对角膜基质损伤修复的调控进行综述,为探索促进角膜基质修复和再生的途径提供新思路,希望帮助临床预防角膜瘢痕的发生。     

转化生长因子β诱导基因在人角膜组织及细胞中的表达

背景 临床研究表明多数角膜营养不良的发病与转化生长因子β诱导(TGFBI)基因突变有关,但其发病的分子机制尚不完全清楚. 目的 研究TGFBI基因在人角膜组织及体外培养的角膜上皮细胞和基质细胞中的表达,为进一步研究角膜营养不良的发病机制奠定基础. 方法 对人角膜上皮细胞和基质细胞进行培养和传代,应用逆转录聚合酶链反应( RT-PCR)法检测TGFBI mRNA在人角膜组织及细胞中的表达,将供体角膜组织制成石蜡包埋切片,利用免疫组织化学法检测TGFBI蛋白在角膜组织、人角膜上皮细胞和角膜基质细胞中的表达,采用免疫荧光技术检测TGFBI蛋白在细胞爬片的表达. 结果 RT-PCR检测显示人角膜组织和基质细胞中在1274 bp处可见TGFBI mRNA的清晰条带,而角膜上皮细胞中亦有TGFBImRNA表达.免疫组织化学检测显示,TGFBI蛋白在人角膜组织中基质细胞的细胞质中呈阳性表达,但人角膜上皮细胞中未见TGFBI蛋白表达.免疫荧光检测技术显示,人角膜基质细胞胞质中TGFBI蛋白呈红色荧光,而角膜上皮细胞未见TGFBI蛋白表达. 结论 TGFBI主要在人角膜基质层表达,而在上皮层几乎不表达,有助于进一步研究TGFBI在角膜营养不良发病机制中的作用.     

Epithelial-stromal interactions: specific stimulation of corneal epithelial cell growth in vitro by a factor(s) from cultured stromal fibroblasts

By using a recently modified method of isolating and culturing rabbit corneal cells, this study investigated the presence of a diffusible substance(s) in stromal fibroblast conditioned medium that stimulated the growth of cultured corneal epithelial cells. The growth stimulation involved initiation of DNA synthesis (assayed by [3H]-thymidine incorporation) and enhanced cell proliferation (quantified by cell counting). Among the three corneal cell types, only fibroblasts (rabbit and human) released the stimulatory substance, which acted only on epithelial cells. The effect of this stromal fibroblast factor (SFF) was observed after an exposure period of less than 16 hr and persisted as long as it was present. Its action was concentration-dependent and was not a result of improvement in the survival of epithelial cells during culture. Both sparse and confluent epithelial cultures were susceptible to SFF. The release of SFF was correlated with the number of fibroblasts in the culture and appeared to be sensitive to the growth condition of the cells. Both the release and action of SFF did not depend on the presence of serum in the culture medium. The factor was heat resistant and insensitive to proteolytic enzymes. From ultrafiltration studies, the size of SFF was estimated to be in the approximate range of 50-1000 daltons. By direct comparison of the stimulatory effect with other previously studied growth promoting agents, it was concluded that SFF was not epidermal growth factor, fibroblast growth factor, putrescine, cyclic AMP, hydrocortisone or acetate. The implication of SFF in the regulation of epithelial growth by endogenous, intercellular mechanisms is discussed.