Effect of basic fibroblast growth factor on cat corneal endothelial cell proliferation

AIM: To investigate the function of basic fibroblast growth factor (bFGF) on cat corneal endothelial cells proliferation. METHODS: Cat corneal endothelial cells were primarily cultured, stimulated with bFGF for different period, the proliferation of cells was assayed by modified tertrozalium salt (MTT) method, and the morphologic changes were observed with inverted phase contrast microscope and transmission electron microscope. RESULTS: At 1, 3 and 5 days after bFGF was added to cat corneal endothelial cells, the result of MTT in 490nm showed significant difference than that in control group, and the difference was most significant in 10ng/mL group. CONCLUSION: bFGF can promote proliferation of cat corneal endothelial cells. 10ng/mL is the relatively most effective dose.     

Inhibition of human scleral fibroblast proliferation with heparin

Proliferation of fibroblasts is a serious problem in ocular trauma and surgical wound healing. Depending on the location of the injury, the growth of fibroblasts can lead to different problems. In glaucoma filtering surgery, fibroblast proliferation may contribute to scar tissue formation and premature wound closure. Fibroblastic growth in proliferative vitreoretinopathy may lead to the formation of preretinal membranes, which can contract, causing retinal detachment. In an effort to find a more effective method of inhibiting ocular fibroblast proliferation, we have investigated the effect of heparin, a sulfated polysaccharide, on the proliferation of fibroblasts obtained from the sclera of donor eyes. Heparin inhibits the incorporation of 3H-thymidine in a dose-dependent manner in the presence of fetal bovine serum (FBS). This inhibition is partially reversed by endothelial cell growth factor (ECGF). The heparin antagonist protamine sulfate causes a reversal of heparin inhibition and, in some instances, a significant increase in 3H-thymidine incorporation compared to serum controls. Heparin was equally effective in inhibiting cell proliferation in control and heparin-protamine sulfate-pretreated medium. These results were apparently unrelated to a direct toxic effect on cells, as a Trypan Blue exclusion assay showed no significant difference in viability when heparin treated cells were compared to control cells. Direct cell counts showed that heparin was effective in inhibiting cell proliferation over a long time period, but only if it was reinstilled every 2 days. Heparin treatment shows promise as a method for controlling fibroblast proliferation in the eye.     

神经生长因子对人角膜上皮细胞增殖的影响

目的研究神经生长因子(nervegrowthfactor,NGF)在体外培养的人角膜上皮细胞中的表达,观察外源性给予NGF对人角膜上皮细胞增殖的作用,进一步探讨NGF在角膜疾病治疗中的意义。方法采用刮片法进行人角膜上皮细胞培养,外源性给予NGF作用于第2代～第3代培养的人角膜上皮细胞,通过四甲基偶氮唑盐(methylthiazolyltetrazolium,MTT)法测定其对细胞增殖的影响,并用流式细胞仪检测角膜上皮细胞中细胞增殖核抗原的表达情况。结果本实验成功培养出人角膜上皮细胞,加入外源性NGF后,培养的人角膜上皮细胞的增殖率较对照组明显增加(P<0.01),流式细胞仪检测到的细胞增殖核抗原的表达也较对照组明显增多(P<0.01)。结论外源性给予NGF对体外培养的人角膜上皮细胞的细胞增殖有明显的促进作用。     

血小板源性生长因子和bFGF促进猫角膜内皮细胞增生的协同作用研究

目的 观察血小板源性生长因子（PDGF-BB）和碱性成纤维细胞生长因子（bFGF）对猫角膜内皮细胞（CECs）增生的影响及其联合应用对细胞增生是否有协同作用。方法 猫CECs原代培养，传1代后，分别在培养液中加不同浓度的PDGF-BB和bFGF，加药后第1、3、5d分别利用MTT法测定在490nm处的吸光值来判断CECs的增生情况；同时应用倒置相差显微镜观察细胞形态，扫描电镜检测细胞超微结构。结果 与对照组比较，加药后第1、3、5d，PDGF-BB和bFGF均能增加培养的猫CECs的数量，最有效质量浓度分别为100ng／ml和10ng／ml，二者联合培养组猫CECs的增生能力更强。结论 PDGF-BB和bFGF可促进猫CECs的增生，二者具有协同作用。     

Effect of human corneal fibroblasts on lymphocyte proliferation in vitro

Cocultivation of human corneal fibroblasts (CFB) with allogeneic peripheral blood mononuclear cells (PBL) induced a minimal lymphocyte proliferative response, even when the fibroblasts had been pre-treated with interferon-gamma (IFN-gamma) and were HLA-DR positive. IFN-gamma-treated CFB did not express detectable HLA-DQ alloantigens. Cocultivation of CFB with PBL in the presence of Concanavalin A (Con A) or Phytohemagglutinin inhibited mitogen-induced lymphocyte proliferation by 40-90% relative to PBL cultured without CFB. Induction of HLA-DR expression on the CFB did not alter their inhibitory properties. Addition of indomethacin to cultures reversed the effect of CFB on Con A responses. However, no difference between proliferative responses to HLA-DR positive or negative CFB was seen in the presence of indomethacin. This weak response to induced Class II alloantigens on CFB suggests that induction of Class II alloantigens on corneal stroma alone may be insufficient to sensitize a recipient for Class II alloantigen-driven corneal graft rejection.     

碱性成纤维细胞生长因子对人晶状体上皮细胞增殖及移行作用的影响

目的 探讨碱性成纤维细胞生长因子（bFGF）对体外培养的人晶状体上皮细胞（HLEC）增殖及移行作用的影响.方法 在无血清培养液培养的HLEC中分别加入不同终浓度的bFCF（0.01、0.1、1、10及100 μg/L）,MTT法测定其促细胞增殖的情况;流式细胞仪分析细胞周期;HLEC损伤愈合模型,观察不同终浓度bFGF处理24 h后HLEC的移行情况.结果 bFGF浓度为0.1、1、10、100 μg/L时,其对HLEC细胞有明显的促增殖作用,与阴性对照组比较差异具有统计学意义（P〈0.01）,100 μg/L作用24 h增殖率最大,达112.78%,作用强度呈浓度时间依赖性.bFGF通过促进细胞周期变化,与阴性对照组比较差异具有统计学意义（P〈0.01）,G0/G1期细胞减少,S期和G2/M期细胞增多,通过促进HLEC由G0向G1的转化来促进细胞的增殖;bFGF浓度1、10、100 μg/L作用24 h.可明显促进HLEC的移行,其移行能力分别为27.21%、154.42%、和238.77%,与阴性对照组相比,差异有显著统计学意义（P〈0.01）.结论 bFGF可促进HLEC的增殖和移行,是HLEC强有力的有丝分裂原和促移行因子.     

酸性成纤维细胞生长因子及其改构体对培养大鼠RGCs存活与生长的影响

目的 观察不同质量浓度aFGF、MaFGF对培养的大鼠视网膜神经节细胞(RGCs)生长的影响,及aFGF的神经保护活性是否依赖促有丝分裂活性.方法 用不同质量浓度的aFGF和MaFGF培养RGCs.免疫细胞化学染色,计数免疫阳性细胞,计算轴突生长细胞百分率,MTT法进行检测.结果 培养第3 d,大部分存活细胞为Thy-1免疫细胞化学反应阳性,5 d、7 d后逐渐减少.实验组存活时间3～4 d;培养第3 d,MTT法见各组A值与对照组比较差异无统计学意义(P＞0.05),培养第5 d、7 d,差异有统计学意义(P＜0.01);培养相同天数不同质量浓度的aFGF及MaFGF比较,差异均有统计学意义(P＜0.05);相同质量浓度的aFGF各组与MaFGF各组比较,差异无统计学意义(P＞0.05).结论 aFGF、MaFGF均能促进培养大鼠RGCs的存活,并延长其存活时间;aFGF保护及促进RGCs存活的作用不依赖其有丝分裂活性.     

神经生长因子对角膜上皮细胞增殖的影响

目的 探讨神经生长因子（nerve growth factor,NGF）对角膜上皮细胞增殖的影响,寻找促进角膜上皮损伤修复的有效方法。方法 在培养兔角膜上皮细胞的培养液中加浓度为5u/ml、50u/ml和500u/ml的NGF,加药后的第3、7天,采用四甲基偶氮唑盐（MTT）法,于酶标仪上测定570nm波长处的吸光度值来观测细胞增殖情况。结论 浓度为50u/ml和500u/ml的NGF对兔角膜上皮细胞增殖有明显促进作用,且两组间有差异,呈剂量依赖性（P＜0.05）,而浓度为5u/ml的NGF促细胞增殖作用不显著（P＞0.05）。结论 外源性NGF对培养的兔角膜上皮细胞增殖有明显促进作用。表明NGF具有临床应用的可能性。     

bFGF对角膜成纤维细胞增殖及胶原合成的影响

目的 探讨碱性成纤维细胞生长因子（bFGF)对兔角膜基质成纤维细胞增殖及胶原合成的影响。方法 （1）分组培养正常和切除前板层的角膜成纤维细胞，分别于细胞贴壁后的3、6、9、12、15天观察细胞生长情况，以及bFGF对细胞增殖的影响，并计数。（2）将兔眼分为对照组和实验组，等面积，等深度切除角膜板层后，对照组滴生理盐水，实验组滴bFGF滴眼液，15天后取材，常规电镜制片，观察并比较角膜基质层胶原合成的状况。结果 （1）bFGF可显著促进正常和受损角膜成纤维细胞的增殖。（2）实验组与对照组相比，前者胶原纤维密度高。纤维结构排列明显有序，规律。结论 bFGF可促进角膜成纤维细胞增殖和胶原的合成，使基质层纤维排列更有序。     

神经生长因子对兔角膜内皮细胞增殖的影响

目的 探讨神经生长因子（NGF）对角膜内皮细胞增殖的影响。方法 在培养兔角膜内皮细胞的培养液中分别添加5U／ml,50U／ml和500u/ml的NGF,加药后第3,7天采用四甲基偶氮唑盐（MTT）法,在酶标仪上测定570nm波长处的吸光度值来观测细胞增殖情况。结果 与对照组比较,加药后第3,7天,3组的NGF对培养的兔角膜内皮细胞增殖均促进作用（P＜0．01）,且呈剂量依赖性。其中50U／ml组及500U／ml组作用强于5U／ml（P＜0．05）,而50U／ml组和500U／ml组比较作用无差异（P＞0．05）。结论 外源性NGF对培养的兔角膜内皮细胞增殖有明显的促进作用。     

Lipid-mediated gene transfer of acidic fibroblast growth factor into human corneal endothelial cells

The aim of this study was to optimize non-viral gene transfer conditions and investigate the effect of fibroblast growth factor-1 (FGF-1) gene transfer on human corneal endothelial cell (HCEC) proliferation. Five non-viral vectors (Lipofectin, DMRIE-C, DAC-30, Effectene, FuGene6) were used to transfect HCEC with plasmids coding for enhanced green fluorescent protein (EGFP) and FGF-1. Transfection efficiency and toxicity (n=6) were quantified and optimized using the EGFP construct by FACS-analysis. Using optimal conditions HCEC were transfected with the FGF-1 plasmid and cell proliferation as well as expression of FGF-1 were determined at days 4 and 7 by counting and western blotting, respectively. Lipofectin (17+/-2.02%) transfected HCEC more successfully than DMRIE-C (11+/-1.46%), Effectene (9+/-0.62%), FuGene (9+/-0.93%) and DAC-30 (7+/-0.59%). Toxicity of the lipids ranged from 2 to 4%. Optimal HCEC proliferation was achieved with DAC-30/FGF-1 (P<0.05), whereas all other vectors did not result in significantly increased cell proliferation. However, all of the transfected cells produced FGF-1 in different amounts as indicated by western blotting. Efficient and almost non-toxic transfer of the FGF-1 gene into HCEC can be successfully achieved by lipid-based techniques. Using optimal conditions significantly increased cell proliferation was independent on gene transfer efficiency. This may indicate that even a low transfection rate is sufficient to produce a concentration of FGF-1 that will have a stimulatory effect on HCECs.     

内皮细胞生长因子和肝素对人脐静脉内皮细胞增殖的影响

目的 研究内皮细胞生长因子(ECGF)和肝素对人脐静脉内皮细胞的促增殖作用，探讨ECGF在新生血管发生中的作用。方法 通过在第4代人脐静脉内皮细胞培养瓶中加入不同浓度ECGF和肝素，应用流式细胞仪分析其细胞周期。结果在ECGF浓度为5—30μg／m1范围内，肝素和ECGF有提高人脐静脉内皮细胞(HUVEC)细胞周期中的S期细胞数、促进细胞的DNA合成的作用。结论 ECGF和肝素对内皮细胞有促进增殖作用。     

Experimental corneal neovascularisation using sucralfate and basic fibroblast growth factor

Purpose: To develop a non-inflammatory model of both acute and chronic angiogenesis in the rabbit cornea using a known directly angiogenic cytokine.
Methods: Pellets made of the slow-release polymer Hydron (polyhydroxyethylmethacrylate) and containing sucralfate and/or basic fibroblast growth factor (basic-FGF) were implanted into rabbit corneas. The neovascular response to the implantation of pellets containing basic-FGF alone, sucralfate alone or a titration of basic-FGF in the presence of a constant amount of sucralfate was measured. The role of inflammation in the neovascular response was also investigated.
Results: The addition of sucralfate to the pellets led to the sustained release of basic-FGF resulting in a predictable and aggressive neovascular response with a low dose of basic-FGF that by itself was unable to elicit neovascularisation. At a dose of 500ng per pellet, approximately one-third of the surface area of the cornea was vascularised within eight days of implantation. Minimal or no vascularisation occurred with the same dose of basic-FGF without sucralfate. While this dose of basic-FGF induced corneal oedema, only minimal inflammation was observed and the response was unaffected by ionising radiation. A less aggressive though still robust neovascular response with no or only minimal oedema was observed when the dose was lowered to 50ng of basic-FGF per pellet. Some induced vessels persisted for more than three months.
Conclusion: This is an inexpensive in vivo model of angiogenesis with the advantages of the neovascularisation being aggressive, predictable, persistent, unassociated with an obvious inflammatory response and induced by the sustained release of an agent known to have a direct stimulatory action on endothelial cells.     

Effects of epidermal growth factor, fibroblast growth factor, and transforming growth factor-beta on corneal cell chemotaxis.

The effects of recombinant basic fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor-beta (TGF-beta) on migration of human and bovine corneal cells were determined using checkerboard analysis in Boyden chambers. EGF, FGF, and TGF-beta each stimulated high levels of chemotactic migration. Each growth factor, however, induced a different dose-response pattern. Migration stimulated by FGF reached a plateau at a concentration between 100 and 200 ng/ml for endothelial, epithelial, and stromal fibroblasts. By contrast, chemotactic responses to EGF peaked between 10 and 50 ng/ml, then decreased at higher concentrations. TGF-beta also stimulated a peak in migration in all three corneal cells, but the peak of migration occurred at an approximately 1000-fold lower concentration (1 pg/ml) than for EGF. Checkerboard analysis demonstrated that FGF and EGF, but not TGF-beta, stimulated chemokinesis of bovine, stromal, and endothelial cells. These results demonstrate that FGF, EGF, and TGF-beta induce migration in pure populations of bovine and human corneal cells and support the concept that these growth factors may play key roles in corneal wound healing by regulating migration of corneal cells.     

重组牛碱性成纤维细胞生长因子对角膜异物剔除术后泪膜的影响

目的 探讨重组牛碱性成纤维细胞生长因子(r-bFGF)滴眼液对角膜异物剔除术后泪膜动态变化的影响.方法 临床病例治疗对照研究.角膜异物剔除术127例(127眼),随机分为治疗组(r-bFGF+氧氟沙星治疗组65例)、对照组(氧氟沙星治疗组,62例).在术前、术后3d、术后5d进行泪膜破裂时间(BUT)、基础泪液分泌试验(SIt)及角膜荧光素染色(FL)检查.结果 两组患者术前上述3项检查结果无差异(BUTt=0.148,P=0.883;SIt t=0.173,P=0.863;FLt=0.730,P=0.467).术后3d两组BUT、FL都有恢复,治疗组恢复明显,两组间差异有统计学意义(BUTt=3.050,P=0.003;FLt=-6.934P=0.000),术后5d两组间BUT差异无统计学意义(t=1.407,P=0.162).术后SIt较术前缩短,术后3d两组间差异有统计学意义(t=-4.825,P=0.000)、术后5d两组间差异无统计学意义(t=1.507,P=0.134).结论 角膜异物剔除术后使用r-bFGF有利于促进角膜上皮的修复,恢复泪膜的稳定性.     

Temporal stimulation of corneal fibroblast wound healing activity by differentiating epithelium in vitro

PURPOSE: To determine whether differentiating corneal epithelium can temporally stimulate fibroblast activity. METHODS: Corneal epithelial cells were cultured to confluence and then stimulated to mature into multilayered epithelia with addition of serum-containing medium. Differentiation was assessed morphologically and immunocytochemically using a monoclonal antibody to cytokeratin 3. At intervals after onset of differentiation serum-free conditioned medium was collected up to 28 days. Preliminary experiments deduced the optimum concentration of conditioned medium to be used for assessing fibroblast activity. Conditioned medium (25% vol/vol) was added to donor-matched corneal fibroblasts in migration chambers, WST-1 reagent proliferation assays, and fibroblast-populated collagen gel contraction assays. Platelet-derived growth factor (PGDF)-AB and interleukin (IL)-1beta in conditioned media were quantified by enzyme-linked immunosorbent assay (ELISA). Fibroblast migration and collagen contraction assays were performed with concentrations of PDGF-AB. RESULTS: Conditioned medium collected from differentiating epithelium stimulated fibroblast migration and collagen gel contraction, with activity peaks occurring with medium collected on day 14 (P < 0.05). No significant difference was detected between fibroblast growth rates in each of the conditioned media. Levels of PDGF-AB increased during epithelial culture up to 22 days (up to approximately 360 pg/ml) with a subsequent decrease by 28 days. IL-1beta inversely correlated with fibroblast activity induced by conditioned medium, with a trough in concentration (2 pg/ml) occurring at 14 days. Both fibroblast migration and collagen contraction were stimulated in a dose-dependent manner by PDGF-AB. CONCLUSIONS: Corneal epithelium is capable of temporally stimulating fibroblast wound-healing characteristics during its differentiation. One of the growth factors potentially involved in this epithelial-stromal interaction is PDGF. This work demonstrated that developing epithelium (possibly similar to repairing epithelium in vivo) regulated fibroblast behavior and may indicate a mechanism of fibroblast recruitment to a wound after epithelial closure.     

细胞因子对角膜成纤维细胞生长的作用

目的,研究细胞团子对角膜成纤维细胞的生长作用,为细胞因子治疗角膜疾病的机制研究提供实验依据。方法：用MTF法测定细胞因子EGF、GTFF－β_1、FN等对角膜成纤维细胞生长的影响作用,结果：EGF对角膜成纤维细胞具有生长调节作用,在0.01~1.0μg/ml浓度下,具有明显促生长作用（与0浓度组比较P＜0．001）,其最佳浓度为0．1μg/ml,但浓度在10.0μg／ml时却对其具有明显抑制作用（与0浓度组比较P＜0005）,TGF－β_1（0.01－10.0ng/ml）、FM（0.01－10．0mg/ml）对角膜成纤维细胞未见明显促生长或抑制作用（P＞0.05）结论：EGF能调节角膜成纤维细胞生长,而TGF、FN对角膜成纤维细胞生长无影响,     

表皮生长因子、碱性成纤维细胞生长因子及5-氟尿嘧啶对培养的人视网膜色素上皮损伤愈合的体外研究

OBJECTIVE: To study the effects of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and 5-fluorouracil (5-Fu) on the cell migration and proliferation. METHODS: Phase-contrast microscopy, cell proliferation assay and wound -closure were used to study the process of cell proliferation and wound healing in a model of human retinal pigment epithelial (RPE) cell damage. RESULTS: It was found that EGF mainly stimulated proliferation and the migration of individual RPE cells from the wound edge, and induced cellular elongation, while bFGF mainly promoted proliferation and spread of the confluent monolayer into the wound defect, and induced enlargement and flattening of cells. But the effects of EGF, bFGF on wound closure were not very significant; 5-Fu inhibited the spread and proliferation of RPE cells, but had no effects on cell migration. CONCLUSIONS: EGF and bFGF have no significant effects on wound-healing, perhaps their effects on cell morphology are very important in cell transformation in proliferative vitreoretinopathy (PVR), and 5-Fu can only be a subsidiary drug to prevent PVR.