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

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

表皮生长因子对人角膜内皮细胞损伤修复的影响

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

重组牛碱性成纤维细胞生长因子对角膜内皮细胞密度低下患者白内障术后角膜内皮的保护作用

目的　研究角膜内皮细胞密度（ECD）低下患者行超声乳化白内障吸除术后应用重组牛碱性成纤维细胞生长因子（re-bFGF）对角膜内皮的保护作用。方法　前瞻性队列研究。选取2019年9月至2022年4月在北京大学第三医院眼科就诊、拟行超声乳化白内障吸除且合并ECD低下的患者80例 (90眼)为研究对象,随机分为两组,试验组41例(45眼),对照组39例(45眼)。术后除常规抗炎治疗外,试验组患眼应用re-bFGF滴眼液每日4次滴眼,对照组患眼应用1 g·L^-1玻璃酸钠滴眼液每日4次滴眼,均使用至术后6个月。对比分析两组患眼术前和术后1个月、3个月、6个月的ECD和中央角膜厚度（CCT）等。结果　术后1个月、3个月、6个月,试验组患眼的ECD和CCT均较术前变化不明显（均为P>0.05）,而对照组患眼的ECD均较术前下降,CCT均较术前增加（均为P<0.05）。术前,试验组和对照组患眼ECD分别为（1120.6±306.1）个·mm^-2、（1040.5±317.3）个·mm^-2 ,CCT分别为（543.1±51.6）μm、（546.8±35.6）μm,两组相比差异均无统计学意义（均为P>0.05）;术后6个月,试验组和对照组患眼ECD分别为（1271.3±288.6）个·mm^-2、（746.5±193.5）个·mm^-2 ,CCT分别为（542.0±55.3）μm、（583.5±45.3）μm,两组相比差异均有统计学意义（均为P<0.05）。术后2眼发生了角膜内皮失代偿,且均发生在对照组。结论　re-bFGF对ECD低下患者行超声乳化白内障吸除术后的角膜内皮有保护作用,可减轻超声乳化手术造成的ECD下降,减少角膜水肿及术后短期角膜内皮失代偿的严重并发症的发生率。     

表皮生长因子对猫角膜内皮细胞DNA合成的影响

目的　观察人表皮生长因子 (EGF)对体外培养的猫角膜内皮细胞损伤愈合速度及DNA合成的影响。方法　猫角膜内皮细胞体外培养传一代融合后 ,定量损伤直径 3mm范围内的细胞 ,培养液中加入 1、10、5 0和 10 0ng/mlEGF ,对照组不加EGF ,损伤后 1、3、5天倒置显微镜下照相转入计算机测量未愈合面积 ,作为判断不同浓度EGF影响角膜内皮细胞损伤愈合速度的指标。培养液中加入3 H 胸腺嘧啶核苷 ( 3 H -TdR) ,应用液体闪烁计数仪测量3 H TdR的掺入量 ,反映不同质量浓度EGF对角膜内皮细胞DNA合成的影响。结果　一定质量浓度的EGF加快猫角膜内皮细胞损伤愈合速度并促进DNA合成 ,显示剂量依赖性 ,10～ 5 0ng/ml时加快愈合速度作用达高峰 ,10ng/ml时促进DNA合成作用达高峰 ,浓度再增高时作用下降。结论　一定质量浓度的EGF能促进体外培养的猫角膜内皮细胞损伤愈合速度 ,并促进其DNA合成。     

生长因子与角膜内皮细胞

角膜内皮细胞是维持角膜透明的关键细胞成分,角膜内皮细胞密度降低和形态异常可导致内皮细胞功能失代偿而降低视力。在伤口愈合过程中,生长因子可增加角膜内皮细胞密度槿刺激内皮细胞再生,促进伤口愈合。肽类生长因子影响着多种细胞生理过程,包括细胞增殖,分化,移行和存活。     

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。     

利用后弹力层撕除术建立兔眼角膜内皮失代偿模型的评估

目的利用后弹力层撕除术建立一种新的角膜内皮失代偿模型以便更好地了解该手术的组织反应。方法根据手术方法的不同将40只新西兰成年兔平均分为4组：角膜内皮刮除组、后弹力层撕除组、后弹力层撕除角膜内皮移植术（DSEK）组及DSEK供体组;右眼为手术眼。每组定期通过角膜内皮活体染色,眼前节照相和UBM至少观察2个月。结果后弹力层撕除组角膜始终保持混浊,角膜内皮刮除组和DSEK组角膜逐渐透明,角膜厚度逐渐降低。活体染色显示角膜后弹力层撕除组术后2个月仍无角膜内皮生长。结论后弹力层撕除术建立的角膜内皮失代偿模型显示了后弹力层撕除后角膜内皮愈合过程,可用于角膜内皮移植的研究。     

上皮生长因子在家兔视网膜色素上皮细胞培养中的作用

本研究对有色家兔视网膜色素上皮（ＲＰＥ）细胞进行体外培养，结果显示：低浓度上皮生长因子ＥＧＦ对ＲＰＥ细胞无刺激作用，而达到适当浓度时对ＲＰＥ细胞有明显刺激作用，但高浓度与适当浓度的作用相同。本文认为适当浓度的ＥＧＦ可促进ＲＰＥ培养细胞生长，缩短传代周期，细胞增殖速度快，细胞数量明显增多，且细胞形态不受影响。ＥＧＦ对体外培养ＲＰＥ细胞的最佳浓度为１０ｎｇ／ｍｌ，这为在体外大量培养ＲＰＥ细胞提供了新的实验数据     

新角膜囊袋法诱生的兔角膜新生血管模型

目的研制一种新的具有一致性、可重复性和易于测量的角膜新生血管(cornealneovascularization,CNV)动物模型,为研究血管抑制剂提供稳定、可靠的体系。方法明胶海棉片(1mm×1mm×1mm)蘸取碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF,500ng)后,再用2%的琼脂糖包裹,将干燥后的小丸植入8只家兔角膜基质层,术后观察4周,墨汁灌注照像,病理组织学检查。结果平均(4.00±0.83)d可见新生血管从角膜缘向植入物伸展,且血管成束生长,新生血管范围局限,10d达最高峰,观察4周未见消退。病理组织学检查,新生血管生长过程中均未见明显的白细胞浸润。结论明胶海绵-bFGF-琼脂糖方法结合角膜囊袋法诱生的兔CNV模型,重复稳定,易于在体观察和测定分析。     

碱性成纤维细胞生长因子和肌肽对中期保存液中角膜的保护作用

目的:探讨碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和肌肽(carnosine)对中期保存角膜内皮细胞(corneal endothelial cell,CEC)活性的影响。方法:角膜中期保存液保存兔角膜,对照组为角膜中期保存液,实验组分为A,B,C组,分别为角膜中期保存液中加入bFGF(20μg/L)、肌肽(5g/L)、bFGF(20μg/L)+肌肽(5g/L),在保存角膜3,7,14d观察角膜大体形态,台盼蓝-茜素红联合染色检测CEC活细胞率,扫描电镜及透射电镜检测细胞超微结构改变。结果:保存3d,各实验组角膜大体形态、CEC活细胞率及超微结构改变与对照组相比无显著差异;保存7,14d,各实验组角膜大体形态、CEC活细胞率及超微结构改变均优于对照组,差异有统计学意义(P<0.01),其中以A,C组CEC活性最好。结论:在角膜中期保存液中加入bFGF和肌肽,均可以提高保存CEC的活性,但与bFGF相比,肌肽的保护作用相对较弱。     

b-FGF,VEGF在碱烧伤大鼠角膜中的表达与新生血管的关系

目的 探讨角膜碱烧伤后,碱性成纤维细胞生长因子（b-FGF）,血管内皮生长因子（VEGF）在大鼠角膜中的表达与角膜新生血管化的关系。方法 采用1mol/L的氢氧化钠溶液烧伤35只Sprague-Dawleg（S-D）大鼠角膜,建立角膜碱烧伤动物模型;碱烧伤后不同时间的形态学分析来评价角膜新生血管的情况,用免疫组织化学染色方法和计算机图像分析系统检测大鼠角膜烧伤后不同时间点b-FGF及VEGF在角膜中的表达。结果 大鼠角膜烧伤后2db-FGF及VEGF开始增高,伤后7d达到高峰,然后2种因子的表达下降,14d后显著下降,碱烧伤后角膜新生血管与两种因子的表达呈明显的平行关系。结论 碱烧伤后,大鼠角膜b-FGF,VEGF的表达与新生血管的形成有相关性,并在其中发挥重要的作用。     

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.     

血小板源性生长因子和碱性成纤维细胞生长因子对兔角膜基质成纤维细胞体外增殖的影响

目的：探讨重组人血小板源性生长因子(rh-PDGF-BB)和重组人碱性成纤维细胞生长因子(rhb-FGF)单独及联合应用对兔角膜基质成纤维细胞(RCF)增殖的影响。方法：采用细胞培养技术及噻唑蓝比色法(MTT)。结果：rhPDGF-BB和rhbFGF对RCF的促增殖作用较对照组有明显提高(P＜0．01)，rhPDGF-BB在10～100μg/L之间时对RCF有较强的促增殖作用(P＜0．01)，并呈剂量效应关系。rhb-FGF在0．1～10μg／L浓度范围内与RCF增殖呈剂量效应关系(P＜0．01)，当浓度增大到100μg／L促增殖作用减弱。rhPDGF-BB和rhb-FGF联合应用时其促增殖作用较两种因子在相同浓度单独应用时有显性差异(P＜0．05)。     

表皮生长因子对家兔角膜内皮损伤修复作用的实验研究

目的 研究表皮生长因子对家兔角膜内皮损伤的修复作用。方法 将溶于透明持酸钠的ＥＧＦ注入角膜内皮已损伤的兔眼前房,对照眼只注入ＮａＨＡ。结果 １０μｇ和５μｇ的ＥＧＦ均可使角膜内皮缺损面积显著缩小,并可使再生的角膜中央内皮细胞密度,六边形细胞出现率显著上升及细胞面积变异系数明显减小。     

Calculated and measured endothelial temperature histories of excised rabbit corneas exposed to infrared radiation

This paper reports calculation of the endothelial and epithelial temperature increases caused by exposing the cornea to radiation from three infrared laser systems: CO₂ holmium and erbium. Data are presented for a variety of exposures and beam diameters. The calculations assume that the lasers are operated with a Gaussian beam intensity profile and the radiation is absorbed according to the Beer-Lambert law. We show that the absorption lengths of some of these laser lines are sufficiently long that the endothelial temperature rise is very close to that of the epithelium. The efficacy of the theoretical model is tested by comparing the calculations with measurements on excised corneas exposed to CO₂ laser radiation. Both a liquid crystal technique and a thermocouple were used to measure the temporal and spatial variations of endothelial temperature. The experimental and theoretical values are in resonable accord which gives added confidence in the calculations for other laser systems and exposures. The results presented here show that infrared laser exposure safety standards should address the possibility of endothelial damage.     

The extracellular matrix and the control of proliferation of corneal endothelial and lens epithelial cells

When rabbit or bovine corneal endothelial cells were plated at low cell density in the presence of high (10%) concentrations of serum, cells maintained on plastic proliferated slowly and after a few days enlarged considerably. If the cultures were exposed to FGF, the cells proliferated actively and, after a week, a confluent monolayer of closely apposed mononucleated cells was formed. In contrast to cells maintained on plastic, cells maintained on an extracellular matrix (ECM) produced by corneal endothelial cells proliferated even faster than cells maintained on plastic and exposed to FGF and no longer required the presence of FGF to reach confluence. Addition of FGF to such cultures did not decrease the mean doubling time, which was already at a minimum (16 hr), nor did it result in a higher final cell density, which was already at a maximum (700–1000 cells/mm²). It can therefore be concluded that, when the proliferation of corneal endothelial cells from two different species is compared, cells maintained on plastic proliferate poorly and FGF is needed in order for the cultures to become confluent. In contrast, when similar cultures are maintained on ECM, they proliferate actively and no longer require FGF in order to become confluent. Similar results were observed when lens epithelial cells were maintained on plastic versus an ECM. Although cells maintained on plastic hardly proliferated, yielding within a few days a population composed of large and binucleated cells, cells plated on an ECM proliferated actively, with an average doubling time for lens epithelial cells of 15 hr during their logarithmic growth phase.The ability of plasma vs. serum to sustain cell proliferation was analyzed using corneal endothelial cells maintained on plastic versus an ECM. It was observed that cultures maintained on plastic proliferate poorly when exposed to plasma. When exposed to serum they proliferate more actively. Nevertheless, in both cases cultures required the presence of FGF in order to become confluent. When similar cultures were maintained on an ECM, they proliferated equally well regardless of whether they were exposed to plasma or serum and no longer required FGF in order to become confluent. One can therefore conclude that the simple change of substrate from plastic to ECM will restore the sensitivity of these cells to agents present in plasma.     

翼状胬肉细胞之间的相互作用

目的探讨翼状胬肉中血管内皮细胞和成纤维细胞之间的相互作用。方法收集翼状胬肉标本,采用血管内皮细胞和成纤维细胞单独培养、条件培养和共同培养的方法构建培养体系,采用ELISA和RT—PCR法检测3种体系培养上清液和细胞中血管内皮细胞生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）蛋白及mRNA含量的变化。结果单独培养、条件培养和共同培养各组培养上清液中VEGF和bFGF的质量浓度增加,差异均有统计学意义（P〈0．05）;细胞单独培养、条件培养和共同培养三者相比,VEGF和bFGF的mRNA表达升高,差异均有统计学意义（P〈0．05）。结论内皮细胞和成纤维细胞有相互上调作用,2种细胞在翼状胬肉的发生发展过程中相互促进。     

糖尿病患者角膜内皮细胞形态学和角膜厚度研究分析

目的：通过对糖尿病患者进行角膜内皮细胞形态学定量分析,评估糖尿病对角膜内皮细胞的影响。
　　方法：应用全自动角膜内皮细胞分析仪对299例360眼进行角膜厚度及内皮细胞形态检测。正常对照组148例175眼,糖尿病患者151例185眼,其中非增殖期组患者92例110眼,增殖期组59例75眼。比较各组患者的中央角膜内皮细胞平均密度、六边形细胞比例、变异系数及角膜厚度,并进行统计学分析。
　　结果：糖尿病组与正常组角膜相比,角膜内皮细胞变异系数及中央角膜厚度增加,中央角膜平均细胞密度以及六边形细胞比例减小,差异有显著性(P<0.05)。糖尿病增殖期组与非增殖期组比较,中央角膜内皮细胞密度降低,角膜内皮细胞变异系数增加及六边形细胞比例减小,差异有显著性(P<0.05),中央角膜厚度增加,但无统计学差异(P>0.05)。
　　结论：糖尿病患者与正常对照者相比,角膜内皮细胞形态结构存在异常,并且随病变程度的加重而加重,尤其以变异系数以及六边形细胞比例变化更为显著,因此糖尿病患者角膜的抗损伤能力下降。     

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.     

碱性成纤维细胞生长因子促进血管内皮细胞增殖作用的实验研究

目的:探讨不同浓度的碱性成纤维细胞生长因子(bFGF)对体外培养的人眼眶内微血管内皮细胞生长的影响。方法:通过不锈钢筛网2次过滤法分离得到眼眶内微血管内皮细胞.将浓度为20,40,80,160μg/L的bFGF分别加入内皮细胞培养液中作为实验组(I-IV)。不加bFGF的作为对照组,分别在24,48,72h,应用MTT法进行细胞计数。结果:用不锈钢筛网两次过滤法成功的分离到内皮细胞。3d内,实验组和对照组的内皮细胞都有不同程度的增殖。培养24h,实验组与对照组比较,P>0.05,无统计学差异;48h,III,IV与对照组及I,II组比较,差异非常显著(P<0.01);72h时,III组与其它实验组及对照组比较,差异非常显著(P<0.01)。结论:一定浓度的bFGF有非常明显的促血管内皮细胞生长的作用,其中以80μg/L的浓度最显著,可能对早期羟基磷灰石眶内植入血管内生延迟有较大的治疗作用。