Partial restoration of the keratocyte phenotype to bovine keratocytes made fibroblastic by serum

PURPOSE: To determine whether keratocytes made fibroblastic in vitro by addition of fetal bovine serum to the medium regain the keratocyte phenotype after culture in serum-free medium. METHODS: Collagenase-isolated keratocytes from bovine corneas were plated in DMEM/F-12 containing 1% horse plasma, to allow cell attachment, and then cultured until day 4 in either DMEM/F-12 alone, to retain the keratocyte phenotype, or in DMEM containing 10% fetal bovine serum, to cause the keratocytes to become fibroblastic. Medium for the fibroblastic cells was replaced on day 4 with serum-free medium, and cells were cultured until day 12. Cell phenotypes were determined on days 4 to 5 and 11 to 12 of culture as follows: (1) by the morphologic appearance on phase-contrast microscopy; (2) by the levels of aldehyde dehydrogenase in the cells, determined by SDS-PAGE and Coomassie blue staining; (3) by the relative synthesis of collagen types I and V, determined by (14)C-proline radiolabeling; (4) by pepsin digestion and analysis of collagen types by SDS-PAGE autoradiography; (5) by relative synthesis of cornea-specific proteoglycan core proteins determined by analysis of chondroitinase- or endo-beta-galactosidase-generated radiolabeled core proteins by SDS-PAGE autoradiography; and (6) by the relative synthesis of keratan sulfate and chondroitin sulfate determined by (35)SO(4) radiolabeling and measuring the sensitivity to endo-beta-galactosidase and chondroitinase ABC. RESULTS: Keratocytes cultured in serum-free medium appeared dendritic and became fibroblastic in appearance when exposed to medium containing serum. Keratocytes and fibroblasts synthesized a similar proportion of collagen types I and V. However, compared with the keratocytes, the fibroblasts possessed no aldehyde dehydrogenase and synthesized significantly higher levels of decorin and significantly lower levels of prostaglandin D synthase (PGDS) and keratan sulfate. Subsequent culture of the fibroblasts in serum-free medium did not restore aldehyde dehydrogenase to keratocyte levels but did restore the cell morphology to a more dendritic appearance and returned the synthesis of decorin, PGDS, and keratan sulfate to keratocyte levels. CONCLUSIONS: The results of these studies indicate that primary cultures of keratocytes made fibroblastic by exposure to serum can return to their keratocyte phenotype in synthesizing extracellular matrix. These results also indicate that the differences in the organization of the collagenous matrix produced by keratocytes and fibroblasts may be related more to the different proteoglycan types than to the collagen types produced.     

Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture.

PURPOSE: To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes. METHODS: Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes. RESULTS: Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum. CONCLUSIONS: A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.     

Modulation of cultured corneal keratocyte phenotype by growth factors/cytokines control in vitro contractility and extracellular matrix contraction

The purpose of this study was to evaluate specific keratocyte phenotypes (keratocyte, fibroblast, myofibroblast) for cell contractility and ability to contract extracellular matrix. Rabbit keratocyte phenotype was modulated by exposure to optimal proliferative doses of IGF-I, IL-1alpha, FGF2, PDGF-AB, and TGFbeta(1). Cells were then evaluated by immunocytochemistry, western blot, collagen gel contraction and LPA stimulation to measure: (1) focal adhesion (FA), fibronectin (FN) and f-actin assembly; (2) expression of alpha-smooth muscle actin (alpha-SMA); (3) ability to contract extracellular matrix and (4) determine contractile ability, respectively. Untreated keratocytes showed no ability to contract collagen matrix. IGF-I and IL-1alpha increased cell proliferation (70.2 and 74.3%, respectively) but did not alter keratocyte phenotype or ability to contract matrix. FGF2 and PDGF induced fibroblast differentiation with FA and FN assembly and significant (p<0.05) extracellular matrix contraction. TGFbeta(1) induced myofibroblast differentiation with prominent FA and FN assembly, expression of alpha-SMA and significantly greater (p<0.05) matrix contraction. Addition of LPA induced actin filament assembly in growth factor starved fibroblasts and myofibroblasts but had no effect on the cultured keratocyte phenotype. We report for the first time that the keratocyte phenotype is non-contractile and that cell quiescence is not a defining characteristic. We further establish that changes in environmental conditions modulate the keratocyte phenotype resulting in physiologically functional differences regarding cell contractility and capacity to contract extracellular matrix.     

Role of the proteasome in TGF-beta signaling in lens epithelial cells

PURPOSE: The durability of the ubiquitin proteasome pathway in the mammalian lens makes this enzyme system a potential contributor to certain cataracts and posterior capsular opacification (PCO). The present study addresses proteasome involvement in TGF-beta induced, cataract-associated gene activation in human lens cells. METHODS: HLE B-3 cells were treated with TGF-beta, in combination with the proteasome inhibitors MG-132 or lactacystin. TGF-beta target gene expression was measured by semiquantitative RT-PCR. Annexin-FITC staining and flow cytometry were used to assess apoptosis levels. Western blot analyses were performed with anti-SnoN and anti-Smad2 antibodies. RESULTS: TGF-beta induced the expression of alpha-smooth muscle actin, fibronectin, and TGF-beta-inducible gene mRNA in HLE B-3 cells and primary cultured human lens cells from donor tissues. TGF-beta also induced a time-dependent decrease in the level of the Smad repressor SnoN. Gamma-glutamyl-cysteine synthetase (gamma-GCS) mRNA levels decreased in the presence of TGF-beta. Proteasome inhibitor cotreatment blocked the induction of alpha-SMA mRNA, the loss of SnoN protein, the decrease in gamma-GCS mRNA, and TGF-beta-induced apoptosis. CONCLUSIONS: The HLE B-3 cell line and primary cultured human lens cells respond similarly to TGF-beta treatments by activating cataract-related gene expression. This response in both of these model systems is blocked by inhibiting the proteasome. This suggests that the proteasome can mediate cataract and PCO-associated changes and therefore is a novel target of medical therapy.     

Identification and characterization of a growth factor secreted by an established cell line of human retinoblastoma maintained in serum-free medium

A growth factor is obtained from a human retinoblastoma cell line (Y-79) growing in culture in the absence of any added serum or hormone. The retinoblastoma derived growth factor (RDGF) is still secreted by the Y-79 cells after they have been growing in a serum-free environment for over 5 months. RDGF stimulates both cell division and thymidine uptake of Swiss mouse 3T3 fibroblasts in media containing 0.2% fetal bovine serum. The thymidine uptake occurs maximally 16–42 hr after addition of RDGF.The factor has an apparent molecular weight of 38,000 as determined by Sephadex G-100 chromatography and is heat and trypsin labile. There appears to be additional material of higher MW which also stimulates thymidine uptake by the 3T3 cells.     

Insulin growth factor and epidermal growth factor trigger mitosis in lenses cultured in a serum-free medium

The mitogenicity of insulin, insulin growth factor (IGF), and epidermal growth factor (EGF) was evaluated on rabbit lenses cultured in medium KEI-4. IGF, the most highly purified of the insulin-like growth factors was a potent mitogen for mammalian lens epithelia cells. IGF and EGF triggered cell proliferation throughout the normally amitotic central and pre-equatorial region of the epithelium. The mitotic response elicited by IGF and EGF was dose dependent, was preceded by DNA synthesis, exceeded that engendered by equimolar insulin, and exhibited a chronology identical to that brought about by crystalline insulin. Lenses cultured in KEI-4 alone or in KEI-4 supplemented with growth hormone, proinsulin, the A and/or B chain of insulin, or MSA, another of the insulin-like growth factors belonging to the somatomedin family, did not show a mitotic response. Simultaneous exposure of the lens to IGF and EGF resulted in an increase in the total number of mitotic figures over that obtained with equimolar concentrations of IGF and EGF. Our results suggest that IGF or other insulin-like growth factors may be capable of regulating cell division in the mammalian lens in vivo. That the lens epithelium responded to IGF and EGF may indicate that lens epithelial cells are subject to multiple hormonal interaction. Since growth factors appear to be cell type specific, information obtained from the rabbit lens epithelium should be useful in delineating the factors and conditions required for the growth of cultured human lens cells.     

Modulation of acute inflammation and keratocyte death by suturing, blood, and amniotic membrane in PRK

PURPOSE: To investigate the role of acute inflammation in keratocyte death, which may influence corneal haze after photorefractive keratectomy (PRK). METHODS: Transepithelial PRK was performed on both eyes of 30 rabbits. Twenty-six rabbits were divided into 4 groups receiving autologous blood, suturing alone, suturing with amniotic membrane graft, or no treatment as the control. Twenty-four hours later, the ablated zone was analyzed for keratocyte death by TdT-dUTP terminal nick-end label (TUNEL) staining and transmission electron microscopy, for polymorphonuclear cell (PMN) infiltration by hematoxylin-eosin staining, and for oxygen radical-induced lipid peroxidation by malondialdehyde immunohistochemistry. The remaining four rabbits were subjected to PRK or mechanical scraping and analyzed immediately or after culturing for 24 hours. RESULTS: Compared with the control group where TUNEL-positive keratocytes were found only in the superficial ablated stroma, blood application or suturing caused more and deeper keratocyte death and PMN infiltration (P: < 0.05). The amniotic membrane graft group had less keratocyte death and PMN than the control or the suture group (P: < 0.05 and P: < 0.01, respectively). There was a strong correlation between keratocyte death and PMN infiltration (P: < 0.01, correlation factor = 0.786). Transmission electron microscopy revealed that the majority of keratocyte death was due to necrosis. Amniotic membrane stroma trapped and prevented PMN infiltration into the stroma. Malondialdehyde-modified antigen was found on the ablated surface and around infiltrated PMN. CONCLUSIONS: Transepithelial PRK causes oxygen radical-mediated lipid peroxidation on the superficial stroma and may contribute to superficial keratocyte death even in the absence of inflammation. Mechanical scraping leads to apoptosis without the participation of inflammation. Keratocyte death by necrosis spreads to the deeper part of the stroma and correlates with additional acute inflammation. Amniotic membrane precludes PMN infiltration and decreases lipid peroxidation and keratocyte death. Future studies are needed to discern whether prevention of inflammation-mediated keratocyte necrosis can reduce unwanted scarring caused by PRK.     

Induction of tissue transglutaminase in the trabecular meshwork by TGF-beta1 and TGF-beta2

PURPOSE: To study whether human trabecular meshwork (HTM) cells are capable of expressing and secreting tissue transglutaminase (tTgase), an enzyme cross-linking extracellular matrix (ECM) proteins, and whether tTgase and synthesis of cross-linked fibronectin are increased after treatment of HTM cells with transforming growth factor (TGF)-beta1 or -beta2. METHODS: Anterior segments of six normal human eyes were stained with antibodies to tTgase. Tissues from three eyes were analyzed for tTgase using Western blot analysis. Monolayer cultures of HTM cells from eyes of five human donors were treated with 1.0 ng/ml TGF-beta1, -beta2, or 5 X 10(-7) M dexamethasone (DEX) for 12 to 96 hours. Induction of tTgase was investigated by Western and Northern blot analysis. External tTgase activity was measured by the ability to form polymerized fibronectin and the incorporation of biotinylated cadaverine into fibronectin. RESULTS: Labeling for tTgase was observed throughout the entire HTM. Cultured HTM cells expressed tTgase intra- and extracellularly. Treatment of cultured HTM cells with TGF-beta1 and -beta2 increased the tTgase mRNA and protein levels, whereas DEX had no effect. TGF-beta-treated HTM cells showed a significant increase in polymerized and unpolymerized fibronectin. Incorporation of biotinylated cadaverine was markedly increased when HTM cells were treated with TGF-beta for 24 hours before seeding. CONCLUSIONS: The enzyme tTgase is expressed in the HTM and is inducible by TGF-beta1 or -beta2 in cultured HTM cells. Extracellular tTgase is able to polymerize fibronectin. Increased levels of TGF-beta2 in the aqueous humor may lead to an increase of tTgase expression and activity in the HTM, causing an increase of irreversibly cross-linked ECM proteins. This mechanism might play a role for the increased outflow resistance seen in glaucomatous eyes.     

Differential expression and regulation of TGF-beta1, TGF-beta2, TGF-beta3, TGF-betaRI, TGF-betaRII and TGF-betaRIII in cultured human corneal, limbal, and conjunctival fibroblasts.

PURPOSE. We have reported that three patterns of cytokine expression are potentially involved between epithelia and fibroblasts of the human ocular surface. The TGF-beta family is a prototypical fibrogenic cytokine responsible for fibroblast activation in wound healing. We investigated how the TGF-beta family is differentially expressed and regulated in cultured human corneal, limbal and conjunctival fibroblasts. METHODS. Human corneal (HCF), limbal (HLF) and conjunctival fibroblast (HJF) were cultured in DMEM-10% FBS until confluence and switched to serum-free DMEM-ITS for 48 h before adding 10 ng/ml of each of eight cytokines for 4 h in three separate experiments. Total RNA was isolated and subjected to Northern hybridization with GAPDH as a control. ELISA was used to determine TGF-beta1 and TGF-beta2 proteins in the media. RESULTS. All three isoforms of TGF-beta and three types of TGF-betaR were expressed by HCF, HLF and HJF. Expression of TGF-beta1 mRNA was strongest and upregulated by the three TGF-betas in all three types of fibroblast. PDGF-BB and TGF-alpha slightly increased TGF-beta1 mRNA. TGF-betas also upregulated TGF-beta3 mRNA in HJF. TGF-betaRI mRNA was the only receptor upregulated by TGF-betas. TGF-betaRII and TGF-betaRIII mRNA were not regulated by all cytokines tested. CONCLUSIONS. TGF-betas auto-induction is the major mechanism upregulating TGF-beta1 expression. Promotion of TGF-beta3 by the TGF-betas may have a special role in HJF. Differential expression and regulation of TGF-betas and TGF-betaRs suggest that each TGF-beta isoform may have specific functions in different ocular surface fibroblasts. No cytokine tested can downregulate TGF-beta1 and the TGF-betaRs.     

TGF-beta1, TGF-beta receptor II and ED-A fibronectin expression in myofibroblast of vitreoretinopathy

PURPOSE: Formation of scarlike epiretinal membranes (ERMs) constitutes potentially the end stage of evolution of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Among various cellular populations, ERMs contain cells with contractile features typical of myofibroblasts. The current study was conducted to investigate the presence of transforming growth factor (TGF)-beta1, TGF-beta receptor II (RII) and ED-A fibronectin (FN), the main inducers of myofibroblastic differentiation in ERMs in PDR and PVR. METHODS: Samples of ERM were obtained from 23 patients during microsurgery for PVR or PDR. Electron microscopy, immunohistochemistry, and confocal microscopy with antibodies recognizing beta-smooth muscle (SM) actin, desmin, TGF-beta1, TGF-beta receptors I and II, and ED-A FN were performed. RESULTS: alpha-SM actin was detected in all ERMs, whereas desmin was present in 50% of the cases. ED-A FN was expressed in all ERMs in close relation with alpha-SM actin-positive myofibroblasts. In addition, TGF-beta1 and TGF-beta R II were always present, TGF-beta RII being expressed in both alpha-SM actin-positive and negative fibroblastic cells. CONCLUSIONS: Myofibroblast accumulation is a key event in ERM-associated traction retinal detachment occurring during PVR and PDR. The current results suggest that the presence of alpha-SM actin-positive myofibroblasts is probably dependent on the concomitant neoexpression of TGF-beta1, TGF-beta RII, and ED-A FN. The results furnish new data on the mechanism of alpha-SM actin stimulation in fibroblasts in a human pathologic setting.     

Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism

BACKGROUND: The success of long-term corneal organ culture is limited by the progressive loss of endothelial cells during culture and the use of culture medium supplemented with fetal calf serum as a possible source of contamination with infectious agents. In this study, we investigated the suitability of a serum-free medium (Endothelial-SFM) to improve preservation conditions for human donor corneas. METHODS: Six pairs of corneas were stored in Minimum Essential Medium (MEM) supplemented with 2% fetal calf serum (FCS) for 8-14 days. One cornea of each pair was then further cultivated in Endothelial-SFM supplemented with 2% FCS or in MEM with 2% FCS, respectively. In a second series of experiments, the endothelial cell density of seven pairs of freshly isolated donor corneas was determined during cultivation in Endothelial-SFM with 2% FCS or serum-free Endothelial-SFM. RESULTS: After precultivation in conventional medium, the endothelial cell density of corneas allocated to cultivation in Endothelial-SFM was 1000-1950 cells/mm2 and that of those subsequently cultured in MEM 1200-2000 cells/mm2. At 9 weeks, cell densities of 900-1500 cells/mm2 were found after cultivation in Endothelial-SFM compared with a total cell loss in MEM. Freshly isolated corneas cultured in Endothelial-SFM with or without FCS supplementation showed a decrease of endothelial cell density of about 20% within the first 2 weeks of storage. During further cultivation cell density remained constant without statistically significant differences between the groups. Glucose consumption of the corneas was higher in Endothelial-SFM than in MEM. Corneas stored in Endothelial-SFM with 2% FCS showed a higher glucose consumption than those preserved in serum-free Endothelial-SFM. CONCLUSION: Organ culture of human donor corneas using the serum-free basal medium Endothelial-SFM is superior to conventional culture conditions because the decrease in endothelial cell density can be ameliorated, the culture period can be prolonged and the risk of transmitting infectious agents via serum can be minimised.     

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.     

兔角膜内皮、上皮及基质细胞体外培养扩增的研究

目的 建立角膜上皮、基质及内皮细胞体外培养扩增的简单稳定的方法，为组织工程化角膜的构建提供种子细胞。方法 内皮细胞与后弹力层在培养基中孵育后消化法获原代细胞，胰酶消化去除表层上皮后取角膜缘，组织块法培养角膜缘上皮细胞，基质细胞应用胶原酶消化法获原代培养，各细胞融合后胰酶消化依次传代培养。结果 原代内皮细胞4—5d融合成单层细胞，可连续传6—7代。上皮细胞1周左右生长融合，连续传3—4代后细胞形态改变。基质细胞接种6—7d后近融合，传代后增殖明显，可连续传10代。结论依据角膜组织特征选择合适的方法体外分离、培养角膜3种细胞成分，可获连续传代扩增的角膜细胞。     

Immunolocalization of TGF-beta1, -beta2, and -beta3, and TGF-beta receptors in human lens capsules with lens implants.

PURPOSE: To establish the alteration in expression pattern of transforming growth factor (TGF)-betas and their receptors during repair of lens capsules after cataract surgery, we immunohistochemically located TGF-beta isoforms and their receptors in human lens capsules before and after cataract surgery. METHODS: Ten post-cataract surgery capsular specimens were obtained during vitrectomy. Three sections of the anterior capsules were obtained during cataract surgery. A whole lens capsular bag immediately after lens extraction was obtained during vitrectomy. Cryosections of these specimens were processed for immunohistochemical analysis for TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta receptor type I (TbetaR-I), type II (TbetaR-II) and type III (TbetaR-III), and were observed under light micros-copy. RESULTS: Lens epithelial cells (LECs) lining the inner surface of the anterior capsules exhibited immunoreactivity for TGF-beta2 and TbetaR-II. Immunoreactivity for TGF-beta1, -beta3, TbetaR-I and TbetaR-III was negative. In the whole capsular bag specimen, equatorial LECs were positive for TGF-beta1 and -beta2, but not for -beta3. In post-cataract surgery specimens, antibodies for each TGF-beta isoform labelled either the LECs or ECM accumulated on the capsules. Post-surgical LECs expressed TbetaR-I and TbetaR-II, and had also TbetaR-III in seven of the nine specimens examined. CONCLUSION: Expression pattern of TGF-beta s in quiescent LECs showed regional heterogeneity. Anterior LECs exhibited TGF-beta2 immunoreactivity, while equatorial LECs were positive for TGF-beta1 and -beta2. Quiescent LECs expressed TbetaR-II. LECs proliferating around IOLs expressed proteins of each TGF-beta isoform and each TbetaR. TGF-beta s were also localized in the ECM on capsules undergoing repair. TGF-beta3, TbetaR-I and TbetaR-III are up-regulated in LECs after cataract surgery.     

准分子激光角膜切削术与磨镶术后兔角膜基质细胞凋亡的研究

目的研究准分子激光角膜切削术(photorefractive keratectomy,PRK)和准分子激光原位角膜磨镶术(laser in situ keratomileusis,LASIK)后不同时间点角膜基质细胞凋亡的特点并探索不同手术方式、不同的矫正度数对角膜基质细胞凋亡水平的影响。方法50只新西兰纯种白兔随机分为A、B两大组,每组25只;A组兔子为PRK组,B组兔子为LASIK组;每组兔子左眼进行-4.0D激光切削,右眼进行-8.0D激光切削。分别在术后4h、24h、1w、4w和3m五个时间点处死兔子,每个时间点每组随即处死5只兔子,取兔角膜制作病理切片,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记法(terminal-deoxynucleotidyl transferase mediated 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的差别。结果在术后4h、24hPRK组角膜基质中凋亡细胞较多,其中PRK-8.0D组的凋亡细胞数较PRK-4.0D组多,其差异有显著性意义(P<0.05);在术后4h、24h两个时间点,PRK-8.0D组与LASIK-8.0D组的角膜基质中的凋亡细胞数进行比较,两者差异有显著性意义(P<0.05);在不同的时间点,LASIK-4.0D组与LASIK-8.0D组TUNEL阳性细胞计数的比较,两者之间差异无显著性意义(P>0.05)。结论LASIK术后角膜基质细胞的凋亡水平较PRK低,凋亡程度与制瓣中的角膜损伤有关,与切削深度无明显相关;PRK术后角膜基质细胞的凋亡水平随切削深度增加而增高。     

Effect of prophylactic and therapeutic mitomycin C on corneal apoptosis, cellular proliferation, haze, and long-term keratocyte density in rabbits

PURPOSE: To determine the mechanism through which topical mitomycin C prevents and treats corneal haze after photorefractive keratectomy (PRK) and to examine the effects of dosage and duration of exposure. METHODS: In 224 New Zealand rabbits, -9.0 diopter PRK with mitomycin C or balanced salt solution was performed. Haze level was graded at the slit-lamp. Rabbits were sacrificed at 4 hours, 24 hours, 4 weeks, or 6 months after surgery and immunohistochemistry was performed with TUNEL assay, Ki67, and alpha-SMA. RESULTS: TUNEL-positive apoptotic cells marginally increased in all mitomycin C groups whereas Ki67-positive mitotic cells decreased significantly following mitomycin C application. A greater decrease in myofibroblasts was noted with prophylactic mitomycin C treatment than therapeutic mitomycin C treatment. There was, however, an anterior stromal acellular zone (approximately 20% of the total stroma) in eyes treated with mitomycin C, which persisted to the maximum follow-up of 6 months. CONCLUSIONS: Mitomycin C treatment induces apoptosis of keratocytes and myofibroblasts, but the predominate effect in inhibiting or treating haze appears to be at the level of blocked replication of keratocytes or other progenitor cells of myofibroblasts. Treatment with 0.002% mitomycin C for 12 seconds to 1 minute appears to be just as effective as higher concentrations for longer duration in the rabbit model. However, a persistent decrease in keratocyte density in the anterior stroma could be a warning sign for future complications and treatment should be reserved for patients with significant risk of developing haze after PRK.     

Stimulation and inhibition of uveal melanoma invasion by HGF,GRO, IL-1alpha and TGF-beta

PURPOSE: To investigate potential factors involved in uveal melanoma migration and invasion in vitro. METHODS: Using a microchemotaxis chamber, the effects were studied of a range of stimulators and inhibitors on a series of 10 primary uveal melanomas and 2 uveal melanoma cell lines, by assessing invasion through an 8- micro m pore membrane, precoated with an extracellular matrix solution. In addition, invasion in response to the effect of cells and conditioned media derived from the liver and other tissues was studied for one uveal melanoma culture, by using double-chambered wells, and invasion was assessed through an 8- micro m pore membrane, precoated with synthetic extracellular matrix. In all instances, invading cells were counted under x400 magnification on the lower surface of the membrane. Levels of invasion were correlated with histopathologic markers of prognosis. RESULTS: Conditioned media and cells derived from other tissues, including the liver, increased cellular invasion of the uveal melanoma cell line studied. For specific regulators, maximum stimulation of invasion was induced by hepatic growth factor (HGF), growth-related oncogene (GRO), and macrophage inflammatory protein (MIP)-1beta, whereas significant inhibition was induced by IL-1alpha, TGF-beta1, and TGF-beta2. CONCLUSIONS: The primary site of metastasis in patients with uveal melanoma is the liver. For the degree of site specificity commonly seen, regulators involved in the process may be expressed at the secondary sites, promoting adhesion, migration, invasion, and proliferation of tumor cells. HGF, GRO, MIP-1beta, IL-1alpha, TGF-beta1, and TGF-beta2 may play a significant role in regulating invasion of uveal melanoma cells.