Effect of cytokines on regulation of the production of transforming growth factor beta-1 in cultured human Tenon's capsule fibroblasts

PURPOSE: Transforming growth factor-beta1 (TGF-beta1) is thought to play a pivotal role in the regulation of the wound healing process after glaucoma filtering surgery. The aim of the present study was to investigate whether platelet-derived growth factor isoforms (PDGF-AA, PDGF-BB), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) modulate the production of latent and/or active TGF-beta1 by cultured human Tenon's capsule fibroblasts (HTF). METHODS: Human Tenon's capsule fibroblasts were seeded at two different densities (30 cells/mm2 and 150 cells/mm2) and stimulated for five days with PDGF-AA, PDGF-BB, bFGF, EGF, IL-1beta and TGF-beta1. Control cells were treated with serum-free medium (WM/F12). The concentrations of latent and active TGF-beta1 in the medium were determined using an immunoassay before and after activation of TGF-beta1 by transient acidification. RESULTS: The concentration of latent TGF-beta1 in conditioned media from HTF seeded at high density (150 cells/mm2) significantly increased after stimulation with 5 ng/ml TGF-beta1 (151.5 +/-41.7 pg/ml) or 10 ng/ml IL-1beta (45.7+/-8.1 pg/ml). The concentration of active TGF-beta1 in conditioned media also significantly increased after stimulation of HTF with 5 ng/ml TGF-beta1 (48.4+/-27.5 pg/ml). CONCLUSIONS: The present results indicate that TGF-beta1 is the most potent inducer of its own synthesis in HTF. Activation of an autocrine TGF-beta1 loop may play a role in the wound healing response after glaucoma filtering surgery.     

Proliferative response of cultured human tenon's capsule fibroblasts to platelet-derived growth factor isoforms

Background: Although platelet-derived growth factor (PDGF) has been thought to be critical in the wound-healing response of Tenon's capsule fibroblasts after glaucoma filtration surgery, no information is currently available concerning the proliferative effect of PDGF isoforms on this cell type. The aim of the present study was to evaluate the proliferative effect of PDGF-AB heterodimer and PDGF-AA and -BB homodimers on cultured human Tenon's capsule fibroblasts. Methods: Human Tenon's capsule fibroblasts, cultured under serum-free conditions, were stimulated with PDGF-AA, -AB and -BB isoforms in concentrations ranging from 1 to 100 ng/ml. Cell numbers were determined on days 1, 3, 5 and 7, using a cell counter. Results: Addition of PDGF-AB and -BB led to a dosedependent increase in cell proliferation. A maximal response (79.9% over control) was obtained after 7 days with 30 ng/ml of PDGF-BB, with an EC₅₀ of 8.9 ng/ml. The maximal increase in cell proliferation caused by PDGF-AB (30 ng/ml) was 54.9%, with an EC₅₀ of 12.5 ng/ml. Stimulation with PDGF-AA revealed a significant effect only with concentrations higher than 30 ng/ml. Conclusion: Our results indicate that PDGF-AB and -BB isoforms are potent stimulators of proliferation of human Tenon's capsule fibroblasts, suggesting that PDGF-AB and -BB isoforms play an important role in the wound-healing response after glaucoma filtration surgery.Presented in part at the annual meeting of the Deutsche Ophthalmologische Gesellschaft, Mannheim, September 1996     

Cytokine regulation of hyaluronate production by human Tenon's capsule fibroblasts

PURPOSE: The high-molecular weight glycosaminoglycan hyaluronate (HA), a component of the extracellular matrix, has been shown to play important roles in many biological processes including cell proliferation, migration and differentiation. In the present study, the effect of cytokines on production of hyaluronate (HA) by human tenon's capsule fibroblasts (HTF) was determined. METHODS: HTF (2(nd) passage) were seeded at a cell density of 30 cells/mm(2) and stimulated by six different cytokines (platelet-derived growth factor (PDGF)-AA, PDGF-BB, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin (IL)-1beta and transforming growth factor (TGF)-beta1). Controls were treated with aliquots of serum-free medium only. Concentrations of HA were determined using a radiometric assay based on the specific binding of HA to HA binding proteins. RESULTS: The concentration of HA in conditioned medium of HTF was significantly increased only after stimulation with PDGF-AA [10 and 100ng/ml], IL-1beta [1 and 10ng/ml] and TGF-beta1 [5ng/ml]. CONCLUSIONS: Production of HA by HTF is regulated by PDGF-AA, IL-1beta and TGF-beta1 and is speculated to be involved in the wound healing reaction after glaucoma filtration surgery.     

Comparative effects of TGF-beta 1 and TGF-beta 2 on extracellular matrix production, proliferation, migration, and collagen contraction of human Tenon's capsule fibroblasts in pseudoexfoliation and primary open-angle glaucoma

PURPOSE: To comparatively investigate the effects of TGF-beta(1) and TGF-beta(2) on extracellular matrix production, proliferation, migration, and collagen contraction of cultured human Tenon's capsule fibroblasts derived from patients with pseudoexfoliation (PEX) syndrome, PEX glaucoma, primary open-angle glaucoma (POAG), and cataract. METHODS: Tenon's capsule fibroblasts obtained from four groups of patients were cultured and stimulated with different concentrations (0.1-10 ng ml(-1)) of TGF-beta(1) or TGF-beta(2) for up to 14 days. Cell proliferation was determined with the WST-1 colorimetric assay, cell migration by using the Transwell assay system, and collagen contraction by computerised analysis of three-dimensional collagen lattices and immunohistochemistry for alpha-smooth muscle actin expression. Expression and synthesis of extracellular matrix components (fibronectin, collagen types I and III) was assessed by enzyme-linked immunosorbent assays, by real-time RT-PCR, and by transmission electron microscopy. RESULTS: Both TGF-beta(1) and TGF-beta(2) in pathophysiological concentrations of 0.1-5 ng ml(-1) stimulated cell proliferation, migration, collagen contraction, alpha-smooth muscle actin expression as well as mRNA expression and secretion of fibronectin, collagen type I, and collagen type III by Tenon's fibroblasts derived from all groups of patients. TGF-beta stimulation occurred in a concentration-dependent manner with different peak activities associated with different fibroblast functions. There was some variability among the different groups of patients with an increased response of cells derived from PEX and POAG patients as compared to cataract patients. Although no statistically significant differences were found between both TGF-beta isoforms, TGF-beta(1) had a more pronounced stimulatory effect on expression and synthesis of extracellular matrix components including the production of elastic microfibrils, particularly in cells derived from patients with PEX syndrome/glaucoma. CONCLUSIONS: These findings suggest a significant contribution of TGF-beta(1) in addition to TGF-beta(2) to the conjunctival scarring process following glaucoma filtration surgery. Due to its pronounced fibrogenic potential, TGF-beta(1) may become another focus for targeting drug therapy, particularly in patients with PEX glaucoma.     

Effect of growth factors on the differentiation of porcine lens epithelial cells

BACKGROUND: Besides cell proliferation, transdifferentiation of lens epithelial cells (LECs) to myofibroblasts is one of the mechanisms of secondary cataract formation. This process is characterized by increased expression of alpha-smooth muscle actin (alpha-SMA). This study investigated the influence of bFGF, TGF-beta2, EGF and IGF-1 on the expression of alpha-SMA in porcine LECs. MATERIALS AND METHODS: Porcine LECs were cultured for 7 days in serum-free medium without or with 1 to 50 ng/ml bFGF, TGF-beta2, EGF or IGF-I. Alpha-SMA was detected immunocytochemically with a mouse monoclonal antibody, and the relative numbers of alpha-SMA-positive cells were calculated. Statistical analysis was performed using Student's unpaired t-test. RESULTS: The ratio of alpha-SMA-positive cells cultured for 7 days in serum-free medium was 36 +/- 11.9 % (mean +/- SD). BFGF significantly reduced this ratio in a dose-dependent manner to 11.2 +/- 7.3 % at a concentration of 50 ng/ml (p < 0.0001). EGF reduced the ratio significantly to 25.1 +/- 15.7 % (p = 0.05) when 50 ng/ml were applied. IGF-1 (10 ng/ml) reduced the relative numbers of transdifferentiated cells to 16.8 +/- 5.8 %, but the reduction was not statistically significant (p = 0.0787). TGF-beta2 (50 ng/ml) slightly increased the relative number of alpha-SMA-positive cells to 44.2 +/- 13.8 %. However, this increase was not significant (p = 0.1202) during a culture period of 7 days. CONCLUSIONS: BFGF and EGF significantly reduced the expression of alpha-SMA by LECs while TGF-beta and IGF-1 had no statistically significant effect. These results suggest that bFGF and EGF do not primarily induce secondary cataract formation by the mechanism of cell transdifferentiation.     

RNA干扰技术抑制体外培养的人眼球筋膜囊成纤维细胞增殖的初步研究

目的 探讨RNA干扰技术对体外培养的人眼球筋膜囊成纤维细胞增殖的抑制作用。方法 采用化学合成的靶向IKK-β的siRNA,转染体外培养的人眼球筋膜囊成纤维细胞,同时以对任何基因均无作用的siRNA作为阴性对照。以逆转录聚合酶链反应（RT—PCR）技术检测IKK-βmRNA表达水平,免疫印迹法检测IKK-β蛋白表达水平。将siRNA的转染浓度分别设为5、10、25、50、100、200nmol／L,进行转染,于转染后48h采用四甲基偶氮唑盐法检测其转染后对成纤维细胞的抑制作用。结果 经转染了靶向IKK-β的siRNA,其成纤维细胞IKK-β的mRNA和蛋白表达水平均受到抑制。各个转染浓度（5、10、25、50、100、200nmol／L）均可抑制成纤维细胞的增殖（P〈0．05）,抑制率分别为10．72％、23．35％、30．84％、51．25％、50．06％、49．63％,50nmol／L时已达最大抑制率。结论 靶向IKK-β的siRNA转染可以降低IKK-β的表达水平,同时对体外培养的人眼球筋膜囊成纤维细胞的增殖具有抑制作用。抗IKK-β的RNA干扰技术可能是抑制抗青光眼术后滤过道瘢痕化的新手段。     

拉坦前列素滴眼液对人眼球筋膜囊成纤维细胞增殖的影响

目的研究拉坦前列素滴眼液对体外培养的人眼球筋膜囊成纤维细胞的增殖有无促进作用。方法体外培养人眼球筋膜囊成纤维细胞。按1∶10比例逐级稀释拉坦前列素滴眼液(0.005%),使药物浓度依次为5.00、0.50、0.05μg/ml直至5×10-8μg/ml。采用噻唑蓝比色法检测在不同浓度药物作用下的细胞增殖情况,并在光学显微镜下观察细胞的形态变化。结果较高浓度组(5.00μg/ml)拉坦前列素滴眼液对人眼球筋膜囊成纤维细胞存在明显的杀伤作用,细胞几乎全部死亡,吸光度(A)值与对照组相比明显下降(P<0.01);低浓度组(<0.05μg/ml)A值较对照组无明显变化(P>0.05)。比较拉坦前列素滴眼液在不同作用时间下的剂量效应曲线,24、48及72h差异均无统计学意义(P>0.05)。结论拉坦前列素滴眼液不能促进体外培养的人眼球筋膜囊成纤维细胞的增殖,在较高浓度时具有细胞毒性,在较低浓度时则对细胞增殖无明显影响。     

The effect of integrin antibodies on the attachment and proliferation of human Tenon's capsule fibroblasts

The integrins are protein heterodimers consisting of noncovalently associated alpha and beta subunits. The adhesive interactions mediated by integrins are necessary for cellular survival and proliferation. In this study we investigated the effects of three different integrin antibodies on the proliferation of human Tenon's capsule fibroblasts in tissue culture. Human Tenon's capsule fibroblasts were cultured into 96 well plates and treated with different concentrations (ranging from 10(-6) to 1 microg ml(-1)) of three different integrin antibodies: human integrin alpha-2 antibody, human integrin alpha-3 antibody and human integrin alpha-5/FnR (fibronectin receptor) antibody. Coulter counter, hexosaminidase, and 3H-thymidine assays were used to determine the inhibitory effects of these integrin antibodies on ocular fibroblasts on days 0 (attachment), 1,3 and 7 following antibody treatment. The concentration of each antibody required to produce a proliferation 50% less than the control (ID50) was calculated for each assay. With respect to attachment, all three antibodies studied displayed some inhibitory activity. All three antibodies also displayed dose-dependent antiproliferative properties, especially at the highest concentration tested after 7 days of exposure. The integrin alpha-2 antibody was the most potent of the inhibitors, followed by the integrin alpha-3 antibody, with the integrin alpha-5 antibody being the least potent antibody tested. In addition, the anti-proliferative activities of the integrin alpha-2 and integrin alpha-3 antibodies increased with increasing incubation time. In conclusion, these integrin antibodies demonstrated some inhibitory effects on the attachment and proliferation of human Tenon's capsule fibroblasts in culture. Further investigation will be required to determine whether integrin antibodies can significantly limit scar formation in vivo without significant toxicity.     

A rabbit corneal epithelial cell line expresses functional platelet-derived growth factor beta-type receptors.

Platelet-derived growth factor (PDGF) is a family of three isoforms termed PDGF-AA, PDGF-AB, or PDGF-BB that induce proliferation in various mesenchymal cells. A rabbit corneal epithelial cell line (SIRC) was chosen to study the effects of the three isoforms of PDGF. These cells express approximately 43,000 PDGF beta-type receptors and less than 2800 alpha-type receptors on their surface. Thus, only PDGF-BB and -AB led to a transient dose-dependent rise in cytosolic free calcium with an effective dose for 50% of cells of 5 ng/ml. The PDGF-induced calcium signal is largely independent of the presence of extracellular calcium.     

精氨酸-甘氨酸-天门冬氨酸-丝氨酸肽对人Tenon囊成纤维细胞的贴壁抑制试验观察

目的：研究精氨酸-甘氨酸-天门冬氨酸-丝氨酸（Arg-Gly-Asp-Ser，RGDS）肽对人Tenon囊成纤维细胞与纤维连接蛋白（FN）粘附、增殖的影响。方法：在传代培养的人Tenon囊成纤维细胞中加入不同浓度的RGDS（1、0.1、0.001g/L），用未贴壁细胞记数法及MTT法测定RGDS肽对人Tenon囊成纤维细胞粘附、增殖的影响。结果：RGDS肽能抑制成纤维细胞在FN上的粘附，呈浓度效应特点，能抑制成纤维细胞的增殖，呈浓度及时间效应特点。结论：在细胞水平上证实RGDS肽可阻止人Tenon囊成纤维细胞对FN的粘附和增殖，具有避免青光眼滤过术后瘢痕化形成的应用前景。     

Effect of beta radiation on proliferating human Tenon''s capsule fibroblasts.

The effects of different doses of beta radiation from a strontium-90 source on the proliferation of human Tenon's capsule fibroblasts were studied. The cultured fibroblasts were exposed to doses of 100, 250, 500, 750, 1000, 1500, and 3000 rads, and cell numbers were counted at days 3, 7, and 14. Treatment inhibited the proliferation of the fibroblasts. At seven days the cells exposed to 3000 rads showed a decrease relative to the starting cell numbers, and at 14 days the cells exposed to 1500 and 3000 rads showed a decrease in cell numbers. The doses of radiation which inhibited cell proliferation more than 50% (at day 7 and 14) and yet did not cause a decrease in the cell population were 500, 750, and 1000 rads. beta Radiation reduces the proliferation of human Tenon's capsule fibroblasts, and at higher doses this effect may be more pronounced one and two weeks after irradiation.     

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.     

Bifunctional Effect of Human HFN—γon Cultured Human Fibroblasts from Tenon‘s Capsule

PURPOSE: To study the effect of human IFN-gamma on in vitro cultured human fibroblasts from Tenon's capsule. MATERIALS AND METHODS: The effect of different concentrations of human IFN-gamma and mitomycin-C (MMC), 5-fluorouracil (5-Fu) on cultured human Tenon's capsule fibroblasts (HTCF) was measured using a MTT [3-(4, 5-dimethylthiazo-2-yl)]-2, 5-diphenyltetrazolium bromide; Thiazolyl blue) colorimetric assay. The results were analyzed using ANOVA of the statistical package for social sciences (SPSS) 9.0 version. The difference was considered to be significant if P < 0.05. RESULTS: The effects of MMC and 5-Fu on the growth of HTCF were negative, while the effects of IFN-gamma on the growth of HTCF were both negative (10(2)-10(4) units/ml in two experiments) and positive (10(6), 10(5), 10 units/ml in two experiments). The inhibition rate of MMC ranged from 5.73% to 46.9%, which was similar to the inhibition rate of 5-Fu ranged from 12.49% to 38.92% (P = 0.351). The inhibition rate of IFN-gamma in two experiments was smaller than MMC and 5-Fu (P < 0.05). CONCLUSION: IFN-gamma has bifunctional effect (both enhancement and inhibition) on proliferation of cultured HTCF. The antiproliferative effect of IFN-gamma was weaker than MMC and 5-Fu. Further study has to be carried out to document the inhibition of scar formation of filtration bleb by IFN-gamma and the molecular mechanisms of its bifunctional effect on HTCF proliferation.     

Effect of the cytostatic agent idarubicin on fibroblasts of the human Tenon''s capsule compared with mitomycin C

BACKGROUND/AIMS: To investigate the in vitro effect of a short time exposure to the anthracycline idarubicin on proliferation, protein synthesis, and motility of human Tenon's capsule fibroblasts in comparison with the antitumour antibiotic mitomycin C. METHODS: After determination of effective concentrations of idarubicin, fibroblasts of the human Tenon's capsule were exposed to idarubicin or mitomycin C at concentrations ranging from 0.1 microg/ml to 1 microg/ml or from 2.5 microg/ml to 250 microg/ml, respectively, for 0.5, 2, or 5 minutes and cultured for 60 days. Cell death by apoptosis caused by idarubicin treatment was confirmed by Hoechst 33258 staining. Further proliferation was explored by cell counting and by (3)H-thymidine uptake. Protein synthesis was measured by (3)H-proline uptake and motility was assessed by agarose droplet motility assay. RESULTS: Idarubicin is able to exert toxicity and to induce apoptosis during a short time exposure of 0.5 minutes at concentrations of 0.3-1 microg/ml resulting in a significant reduction in cell number compared with the control after 60 days. For mitomycin C, higher concentrations and longer expositions were necessary. Even after treatment with 1 microg/ml idarubicin or 250 microg/ml mitomycin C a few cells were able to incorporate (3)H-thymidine. (3)H-proline uptake up to 10 days after exposure to 0.3 microg/ml idarubicin was found not to be decreased. Cell motility was reduced after treatment with 1 microg/ml idarubicin for 5 minutes or with 250 microg/ml mitomycin C for 2 or 5 minutes. For low mitomycin C concentrations, an increase in motility was found during the first 10 days. CONCLUSION: Idarubicin reduces proliferation of human Tenons's capsule fibroblasts after incubation for 0.5 minutes at concentrations as low as 0.3-1 microg/ml. In comparison, mitomycin C requires longer exposure times and higher doses for equal results. Therefore, idarubicin may be useful in the prevention of glaucoma filtering surgery failure.     

Anti-allergic drugs inhibit the proliferation of human Tenon's capsule fibroblast and maintain the experimental filtering blebs on rabbit eyes

PURPOSE: To examine whether tranilast and ketotifen fumarate inhibit the growth of human Tenon's capsule fibroblasts and maintain experimental filtering blebs on rabbit eyes. METHODS: Human Tenon's capsule fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and growth was measured with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and direct counting of cell number. Production of collagen and transforming growth factor-beta 1 (TGF-beta 1) was measured with corresponding enzyme-linked immunosorbent assay (ELISA). In experimental filtering surgery performed on rabbit eyes, the effects of the topical drugs on intraocular pressure and on maintenance of the filtering blebs were observed. RESULTS: Both tranilast and ketotifen inhibited the growth of the fibroblasts and half-inhibitory concentrations were 200 microM and 70 microM, respectively. Low concentration (10 microM) of tranilast and ketotifen decreased the synthesis of collagen but neither drug had any obvious effect on TGF-beta 1 production. Both drugs prolonged the life of the experimental filtering bleb significantly [12.5 +/- 3.2 (mean +/- standard deviation) days, 13.9 +/- 3.4 days, respectively; control, 10.5 +/- 2.4 days]. CONCLUSION: Tranilast and ketotifen inhibited the growth of human Tenon's capsule proliferation and are capable of maintaining experimental filtering blebs in rabbit eyes.     

CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-beta1

PURPOSE: To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-beta1-induced myofibroblast differentiation. METHODS: Collagenase-isolated keratocytes were seeded and subcultured on plastic or amniotic membrane matrix (AM) in DMEM, with or without 10% FBS, in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS) with 10, 100, and 1000 pg/mL TGF-beta1 or in DMEM with 1% FBS and 10 ng/mL TGF-beta1. Protein expression of CD34 and alpha-smooth muscle actin (alpha-SMA) was measured by Western blot and immunostaining. RESULTS: Keratocytes, expressing CD34 in normal human corneas, continued to express CD34 when cultured on AM in serum-containing medium and on plastic in serum-free medium, but expression was lost on plastic in serum-containing medium. In serum-containing medium, expression of CD34, but not alpha-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed alpha-SMA without CD34 when continuously passaged on plastic. Expression of alpha-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas alpha-SMA expression was upregulated when cells were subcultured on plastic. In serum-free medium, CD34 expression was maintained by cells treated with 10 pg/mL TGF-beta1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became alpha-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-beta1, but failed to do so if cultured on AM, even for 7 days. CONCLUSIONS: These findings indicate that CD34 is expressed by human keratocytes in vivo and in vitro. Myofibroblast differentiation promoted by TGF-beta1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-beta signaling.     

Role of chymase on growth of cultured canine Tenon's capsule fibroblasts and scarring in a canine conjunctival flap model

Chymase is a chymotrypsin-like serine protease contained in the secretory granules of mast cells. Recently, we reported that chymase activity and the number of chymase-positive mast cells in conjunctival tissues were significantly increased during the wound healing process in a hamster model of glaucoma surgery. However, it has been unclear the role of chymase on conjunctival scarring. In the present study, we evaluated the effect of dog chymase on cell proliferation of fibroblasts established from canine Tenon's capsule and the effect of a chymase inhibitor on scarring in a canine conjunctival flap model. After a fibroblast cell culture was established from canine Tenon's capsules, the fibroblasts were incubated in the presence of dog chymase (5-20 ng ml(-1)). Cell proliferation was evaluated by bromodeoxyuridine incorporation. In a canine conjunctival flap model, a sponge treated with a chymase inhibitor, Suc-Val-Pro-Phe(P)(OPh)(2), or placebo was placed in between the conjunctiva and sclera and the conjunctival incision was closed. One week after the surgery, adhesion degree was assessed, and chymase activities in the conjunctival lesion and in the areas of the conjunctiva and sclera were measured. In cultured canine Tenon's capsule fibroblasts, dog chymase significantly increased cell proliferation, and this chymase-dependent proliferation was completely suppressed by the chymase inhibitor. In the canine surgical model, chymase activity in placebo-treated eyes was significantly increased compared to control eyes, while it was significantly decreased by treatment with the chymase inhibitor. Scores for adhesion degree in the chymase inhibitor-treated eyes were significantly decreased in comparison with those in placebo-treated eyes. The conjunctival area in the chymase inhibitor-treated eyes was also suppressed to 52.6% compared with that in placebo-treated treated eyes. In conclusion, chymase stimulates proliferation of fibroblasts derived from canine Tenon's capsule and chymase may play an important role in scarring after glaucoma surgery.     

体外转染γ-干扰素基因抑制人眼球筋膜囊成纤维细胞的研究

目的探讨阳离子脂质体介导的γ-干扰素(IFN-γ)基因对体外培养的人眼球筋膜囊成纤维细胞增殖的抑制作用。方法采用脂质体介导的方法,以质粒pcDNA3 IFN-γ基因转染体外培养的人眼球筋膜囊成纤维细胞,同时以空载体重组质粒pcDNA3作为对照,G418筛选抗性克隆并扩增。以RT-PCR、免疫组化及流式细胞仪间接免疫荧光法检测IFN-γ基因的表达,应用流式细胞仪进行细胞周期分析,采用四甲基偶氮唑盐法检测IFN-γ基因对成纤维细胞的作用。结果转染pcDNA3 IFN-γ基因的成纤维细胞在转录水平和蛋白水平表达IFN-γ基因,转染的IFN-γ基因可明显抑制成纤维细胞的增殖。结论转染的IFN-γ基因对体外培养的人眼球筋膜囊成纤维细胞的增殖具有抑制作用。