Effect of recombinant human platelet-derived growth factor B on cat corneal endothelial cell viability mediated by adeno-associated virus

AIM: To transduce recombinant human platelet-derived growth factor B(PDGF-B) gene adeno-associated virus(AAV) to in vitro cultured cat corneal endothelial cell (CEC) and observe the effect of the expressed PDGF-BB protein on the viability of cat CEC. METHODS: Cat cornea endothelium was torn under microscope and rapidly cultivated in DMEM to form single layer CEC and the passage 2 endothelial cells were used in this study. The recombinant human PDGF-B gene AAV was constructed and transduced into cat CEC directly. Three groups were as following: blank control group, AAV control group and recombinant AAV group. At 24 hours, 48 hours, and 5 days after transduction, total RNA was extracted from the CEC by Trizol and the expression of PDGF-B gene was detected by fluorescence quantitative polymerase chain reaction. Viability of the transduced CEC was detected at 48 hours after transduction by MTT assay. Cell morphology was observed under inverted phase contrast microscope. RESULTS: With the torn endothelium culture technique, we rapidly got single layer cat CEC. At 24 hours, 48 hours and 5 days after transduction, fluorescence quantitative polymerase chain reaction showed there was no significant difference of the expressed PDGF-B gene mRNA between blank control group and AAV control group (P>0.05). In contrast, there were significant differences between two control groups and recombinant AAV group (P<0.05). MTT assay showed that in recombinant AAV group, the expressed PDGF-BB protein could promote the viability of cat CEC. Morphology observation showed at 48 hours after transduction, cells in CEC-AAV-PDGF-B group proliferated into bigger scales in regular triangle to hexagon shape with distinct boundary, while the number of cells was significantly less in the two control groups. CONCLUSION: The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CECs, which promotes the viability and proliferation of cells.     

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.     

脂质体介导兔角膜内皮细胞基因转染的观察

目的观察阳离子脂质体Lipofectamine2000能否介导目的基因转移至兔角膜内皮细胞内及脂质体潜在的毒副作用。方法RT-PCR检测特异性胶原Ⅷ进行细胞鉴定,通过体外细胞转染实验优化阳离子脂质体与质粒DNA的浓度和比例,选择Lipo-fectamine^TM 2000/pEGFP-N1比值3∶1,脂质体体积分别为0μL、6μL、9μL、12μL转染兔角膜内皮细胞,荧光显微镜观察目的基因的表达,透射电镜观察细胞超微结构。结果RT-PCR扩增得到单一产物,与预期的扩增序列大小完全一致,角膜内皮细胞内可见脂质体介导的绿色荧光蛋白表达,Lipofectamine^TM 2000浓度能显著影响其对兔角膜内皮细胞的转染效率,存在最佳浓度,在35mm培养皿中,3μg DNA与9μL脂质体转染效率最高,透射电镜显示12μL脂质体可导致细胞器出现肿胀、崩解现象。结论阳离子脂质体可有效介导目的基因转移至角膜内皮细胞内,在35mm培养皿中,12μL脂质体对角膜内皮细胞有一定的毒副作用。     

氮酮促进脂质体介导质粒DNA转染兔角膜内皮细胞及毒性的实验研究

目的探讨氮酮(AZONE)促进脂质体介导质粒DNA转染兔角膜内皮细胞的作用及其对细胞活性的影响。方法采用细胞培养方法,利用绿色荧光蛋白基因做报告基因,配制不同转染液,脂质体联合pEGFP-N1、氮酮联合脂质体及pEGFP-N1、氮酮联合pEGFP-N1、单纯pEGFP-N1、单纯脂质体、单纯氮酮,分别处理兔角膜内皮细胞,荧光显微镜观察细胞中绿色荧光蛋白的表达。用RT-PCR方法检测EGFPmRNA表达情况。采用MTT法研究不同转染液对细胞活性的影响。结果单纯氮酮、单纯脂质体及对照组处理的细胞不表达绿色荧光蛋白。氮酮联合脂质体及pEGFP2N1对兔角膜内皮细胞的转染效率为42.6%,脂质体联合pEGFP2N1组的转染效率为22.4%,氮酮联合PEGFP-N1组的转染效率为7.2%,单组质粒组的转染效率为1%。氮酮加脂质体加PEGFP-N1组转染效率高于其他转染组(P<0.01)。各组转染液对角膜内皮细胞活性无影响。结论氮酮可促进脂质体介导质粒DNA转染兔角膜内皮细胞,在有效的转染浓度下对细胞无明显毒性,有望成为促进角膜内皮细胞基因转染的新型试剂。     

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。     

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。     

脂质体介导外源基因体外转染兔角膜内皮细胞条件的优化

目的:观察阳离子脂质体LipofectamineTM 2000是否能介导目的基因转移至兔角膜内皮细胞,并优化转染条件。方法:体外培养兔眼角膜内皮细胞,以绿色荧光蛋白(GFP)为报告基因,通过体外细胞转染实验,用不同的细胞融合度、脂质体浓度、脂质体与质粒的比例、转染时间优化转染条件,荧光显微镜观察目的基因的表达。结果:角膜内皮细胞内可见脂质体介导的绿色荧光蛋白表达,LipofectamineTM在细胞融合度为80%~90%,脂质体/DNA为3L/g,脂质体的量为1.8μL,转染时间为5h时,转染效率最高。结论:阳离子脂质体可有效介导目的基因转移至角膜内皮细胞内,GFP可作为报告基因优化脂质体转染条件。     

人血小板源性生长因子B链基因的克隆表达及其对猫角膜内皮细胞体外增殖的影响

目的构建血小板源性生长因子B（PDGF-B）原核表达载体,表达足量的PDGF-BB蛋白,作用于体外培养的猫角膜内皮细胞,观察角膜内皮细胞增殖情况。方法从健康剖宫产妇胎盘组织中提取总RNA,通过逆转录聚合酶链反应（RT-PCR）,扩增出PDGF-BcDNA,将其克隆至含T7启动子的质粒pET-28a（＋）中,构建表达质粒pET-PDGF-B,转化大肠杆菌BL21（DE3）,获得表达菌株BL-PDGF-B,进行PDGF-BB蛋白的表达,以Ni^2＋-NTA树脂对表达蛋白纯化、复性;将得到的PDGF-BB蛋白作用于体外培养的猫角膜内皮细胞,用MTY法分析其对细胞增殖的影响;通过倒置相差显微镜、透射电镜观察细胞形态变化以及细胞内部超微结构。结果经基因测序证明,成功构建出pET-PDGF-B质粒;凝胶自动扫描分析表明,PDGF-BB在BL21（DE3）大肠杆菌中得到了高效表达,表达的PDGF-B单体蛋白经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）,显示了1条特异蛋白带,相对分子质量约为27000;MTY法证明所表达的PDGF-BB蛋白可促进体外培养的猫角膜内皮细胞增殖。结论pET-PDGF-B原核表达载体的成功构建和PDGF-BB蛋白制备纯化为生产活性PDGF-BB蛋白及其进一步功能研究奠定了基础。PDGF-BB蛋白具有促进猫角膜内皮细胞增殖的作用。（中华眼科杂志,2006,42：415-419）     

人脐静脉内皮细胞移植治疗角膜内皮功能衰竭的动物实验研究

目的：探讨异种脱细胞角膜基质为载体培养人脐静脉内皮细胞（HUVEC）进行后板层角膜移植（PLEK）治疗角膜内皮衰竭的可行性。方法：新西兰白兔30只,随机分为3组,实验组、基质组和对照组,每组10只,术中去除角膜内皮细胞,建立角膜内皮衰竭动物模型,实验组和基质组行后板层角膜移植术,对照组仅去除角膜后板层组织,不进行移植。术后观察3mo,对三组角膜的水肿混浊程度和中央角膜厚度进行统计学分析。结果：术后7d,实验组角膜水肿程度较基质组和对照组明显减轻,透明度增加。术后3mo时,实验组内皮细胞密度为2 026.4±129.3个/mm2,中央角膜厚度平均为505.2±25.4μm,基质组中央角膜厚度平均为1 535.6±114.5μm,而对照组为1 493.5±70.2μm。结论：实验以异种脱细胞角膜基质为载体培养人脐静脉内皮细胞,行后板层角膜移植术,治疗角膜内皮衰竭取得了初步成功。移植的人脐静脉内皮细胞能够在活体上成活,并具有一定的角膜内皮细胞的生物学功能,维持角膜透明,为临床上治疗角膜内皮疾病提供了新的思路和方法。     

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.     

Effects of ranibizumab on human corneal endothelial cells

PurposeThis study aims to evaluate corneal endothelial changes occurring over a 3-month period after intravitreal injections of ranibizumab in patients with wet age-related macular degeneration.MethodsThis is a prospective case series. A total of 29 patients (29 eyes) received a 0.5-mg intravitreal injection of ranibizumab. Specular microscopy, including measurement of central corneal thickness and endothelial cell count, was performed on each patient prior to and after completing three intravitreal injections.ResultsAll patients received three intravitreal injections and were followed up for a mean of 3 months. There was no significant change in corneal thickness (p = 0.32) or endothelial cell density (p = 0.38) after ranibizumab injections.ConclusionIntravitreal ranibizumab injections (0.5 mg) have no harmful effects on corneal endothelial cells.     

羊膜培养液体外抑制兔角膜上皮细胞血管内皮生长因子的表达

目的:探讨羊膜培养液对角膜上皮细胞中血管内皮细胞生长因子(vascula rendothelial growth factor,VEGF)表达的影响。方法:刮除并收集新鲜兔角膜上皮细胞,传代培养接种于35mm培养皿及自制的羊膜培养皿上。实验分为4组,Ⅰ组(对照组):无血清的DMEM培养液,Ⅱ组:去上皮的羊膜培养液,Ⅲ组:未去上皮的羊膜培养液,Ⅳ组:将细胞直接接种于无上皮羊膜。作用48h后用Trizol法提取各组样本的总RNA,进行RT-PCR一步法反应检测各组VEGF mRNA表达并与β-actin比较。结果:正常角膜上皮细胞中有VEGF基因表达,在Ⅲ,Ⅳ组中表达受到明显抑制(P<0.01,n=5)。结论:羊膜培养液明显抑制VEGF mRNA在角膜上皮细胞中的表达。     

Magnetic attraction of iron-endocytosed corneal endothelial cells to Descemet's membrane

PURPOSE: To evaluate the feasibility of a novel method of magnetic attraction of iron-endocytosed corneal endothelial cells to Descemet's membrane. METHODS: Cultured rabbit corneal endothelial cells (RCEC) were exposed to spherical iron powder at various concentration ranging 0-100 micro moll(-1). After 24hr, the cell density and morphology were evaluated. RCEC that had been exposed to spherical iron powder (RCEC-iron), were trypsinized and poured onto a culture dish where a neodium magnet was fixated paracentrally. After 24hr, the cell density was measured at the areas with and without a magnet. Rabbits' corneas were cryo-injuried to detach corneal endothelial cells and 1x10(5)/200 micro l RCEC-iron were injected into the anterior chamber. Neogium magnet was fixed on the lid for 24hr to attract RCEC to Descemet's membrane. Each operated eye was observed up to 2 months after the injury. RCEC group (rabbits with cryo-injury and injection of normal cultured RCEC) and cryo group (rabbits with cryo-injury but without injection of RCEC) served as controls. RESULTS: The RCEC-iron density on the dish decreased in the medium containing iron powder of 10 micro moll(-1) or more. When RCEC had been exposed to iron powder of between 5 and 10 micro moll(-1), the ratio of RCEC in the field with a magnet to RCEC in the field without a magnet increased. In the RCEC-iron group, the mean corneal thickness gradually decreased and was significantly less than in the other two groups at 2, 4, and 8 weeks after the cell injection. Fluorescein microscopic examination showed a monolayer of DiI-labelled cells on the Descemet's membrane. CONCLUSION: Magnetic attachment of iron-endocytosed corneal endothelial cells to Descemet's nembrane can be a method of choice for corneal endothelial decompensation.     

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。     

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     

Effects of corneal stromal cell- and bone marrow-derived endothelial progenitor cell-conditioned media on the proliferation of corneal endothelial cells

AIM: To explore the effects of conditioned media on the proliferation of corneal endothelial cells (CECs) and to compare the efficiency of different conditioned media (CM). METHODS: Rat CECs, corneal stromal cells (CSCs), bone marrow-derived endothelial progenitor cells (BEPCs), and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated and cultured in vitro. CM was collected from CSCs, BEPCs, and BMSCs. CECs were cultivated in different culture media. Cell morphology was recorded, and gene and protein expression were analyzed. RESULTS: After grown in CM for 5d, CECs in each experimental group remained polygonal, in a cobblestone-like monolayer arrangement. Immunocytofluorescence revealed positive expression of Na+/K+-ATP, aquaporin 1 (AQP1), and zonula occludens 1 (ZO-1). Based on quantitative polymerase chain reaction (qPCR) analysis, Na+/K+-ATP expression in CSC-CM was notably upregulated by 1.3-fold (±0.036) (P<0.05, n=3). The expression levels of ZO-1, neuron specific enolase (NSE), Vimentin, paired homebox 6 (PAX6), and procollagen type Ⅷ (COL8A1) were notably upregulated in each experimental group. Each CM had a positive effect on CEC proliferation, and CSC-CM had the strongest effect on proliferation. CONCLUSION: CSC-CM, BEPC-CM, and BMSC-CM not only stimulated the proliferation of CECs, but also maintained the characteristic differentiated phenotypes necessary for endothelial functions. CSC-CM had the most notable effect on CEC proliferation.     

人表皮生长因子对体外培养兔角膜内皮细胞影响的实验研究

目的：明确人表皮生长因子（ｈｕｍａｎ Ｅｐｉｄｅｒｍａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ,ｈＥＧＦ）对角膜内皮细胞的影响。方法：采用体外培养的兔角膜内皮细胞,观察接种后不同时点ｈＥＧＦ对其生长状态的影响;兔角膜片内皮损伤模型,离体培养后行Ｈ－Ｅ染色和Ｈ＾３－ＴｄＲ掺入放射自显影,观察ｈＥＧＦ对其修复的影响。结果：兔角膜内皮细胞体外培养ｈＥＧＦ组细胞数高于对照组,并使细胞形态产生纺锤形改变;角膜内皮细胞     

Transplantation of corneas reconstructed with cultured adult human corneal endothelial cells in nude rats

PURPOSE: The feasibility of corneal reconstruction with cultured adult human corneal endothelial cells (HCEC) was examined in a nude rat model. METHODS: Endothelial cells were removed from the corneas of Lewis rats using a sterile cotton swab. Cultured adult HCEC labelled with a fluorescent marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) were seeded onto the denuded Descemet's membrane. Then the corneas were centrifuged, incubated for 2 days, and transplanted into the eyes of nude rats using the penetrating keratoplasty technique (HCEC group). Control nude received corneas denuded of endothelium and without HCEC. The operated eyes were observed for 28 days after transplantation, and then were subjected to histological and fluorescein microscopic examination. RESULTS: The mean corneal thickness was significantly smaller in the HCEC group than in the control group throughout the observation period. The corneal endothelial cell density of the grafts at 28 days postoperatively ranged from 2425 to 3250 cells mm(-2) (mean+/-sd, 2744+/-337 cells mm(-2)). Fluorescein microscopy at 28 days after surgery showed numerous DiI-labelled cells on the posterior corneal surface in the HCEC group. Frozen sections showed a monolayer of DiI-labelled cells on Descemet's membrane. CONCLUSIONS: Cultured adult HCEC function well and maintain corneal transparency for 1 month after transplantation in nude rats.     

恒河猴血管内皮细胞移植替代角膜内皮细胞后房水血管内皮生长因子水平的变化

目的探索恒河猴血管内皮细胞移植替代角膜内皮细胞后房水血管内皮生长因子(vascular endothelial growth factor,VEGF)浓度的变化。方法实验组将体外培养增殖的恒河猴视网膜-脉络膜血管内皮细胞通过离心沉淀法移植到撕除后弹力层的恒河猴角膜内表面;对照组撕除角膜内皮层后,不做任何处理,直接原位缝合角膜植片。分别于术前及术后1周、2周、3周、4周、6周、8周、12周抽取房水,通过ELISA方法检测其中VEGF浓度,并进行统计分析。结果实验组和对照组术后1周时VEGF浓度差异无统计学意义(P>0.05),术后2周、3周、4周时实验组VEGF浓度分别为(374.74±3.30)ng·L-1、(419.06±1.39)ng·L-1、(481.83±3.36)ng·L-1,同时间点对照组分别为(271.11±1.12)ng·L-1、(345.18±3.27)ng·L-1、(380.33±5.03)ng·L-1,差异均具有统计学意义(均为P<0.05),相应时间点实验组显著高于对照组。实验组术后1周、2周、3周、4周VEGF浓度的差异均有统计学意义(均为P<0.05),各时间点与术前(128.75±3.83)ng·L-1差异均有统计学意义(均为P<0.05)。对照组术后各时间点VEGF浓度与术前差异均有统计学意义(均为P<0.05)。结论血管内皮细胞移植到角膜内表面后,房水VEGF浓度在术后4周内持续升高,之后稳定在一较高水平。     

角膜内皮细胞培养的研究进展

角膜内皮功能失代偿引起的失明可通过角膜移植来治疗.人们通过体外培养角膜内皮细胞并使其增殖,进行角膜内皮细胞移植和角膜组织工程学研究.目前常用的分离角膜内皮细胞的方法是从供体上撕除后弹力层和角膜内皮层,利用酶消化作用使角膜内皮细胞分离.常用的培养基为基础培养基加各种生长因子.这些生长因子在一定程度上促进细胞增殖,但培养的角膜内皮细胞长期传代后仍难以维持正常的细胞形态和功能.基因转染技术建立了永生化角膜内皮细胞系,有关细胞周期、信号通路和离子通道的研究成果也促进了角膜内皮细胞体外培养的研究进展.本文综述了角膜内皮细胞体外培养各个环节的最近进展.