Ⅲ型和Ⅵ型膠原在正常角膜和圆锥角膜中的表达

目的 观察正常人及圆锥角膜中胶原Ⅲ、Ⅵ型胶原的表达,探讨Ⅲ、Ⅵ型胶原在圆锥角膜病变中的变化。方法 采用免疫组织化学-SABC法检测角膜组织中Ⅲ、Ⅵ型胶原的表达。结果 Ⅲ型胶原在正常角膜和圆锥角膜的上皮层、实质层及内皮层均有阳性表达,但圆锥角膜比正常角膜的表达弱,而圆锥角膜基质斑块状瘢痕区可见Ⅲ型胶原呈强阳性表达;正常角膜和圆锥角膜组织中在Bowman's层、Descemet's层和基质层均可检测到Ⅵ型胶原表达,但圆锥角膜中Ⅵ型胶原表达减低,特别在圆锥角膜的疤痕区内,Ⅵ型胶原明显降低。结论 Ⅲ、Ⅵ型胶原的异常表达会导致角膜基质纤维间隙的稳定性降低,改变结缔组织中胶原束的组成,最终影响角膜的正常形态。     

I型和II 型胶原在正常角膜和圆锥角膜中的表达

目的　观察正常人及圆锥角膜中 I、II型的表达 ,探讨胶原 I、II型在圆锥角膜病变中的变化。方法　采用免疫组织化学 - SA BC法检测角膜组织中胶原 I、II型胶原的表达。结果　胶原 I型在正常角膜和圆锥角膜的上皮层、实质层及内皮层均有阳性表达 ,但圆锥角膜比正常角膜的表达弱 ;而胶原 II型在正常角膜和圆锥角膜组织的基质层和 Bow m an层均可检测到 ,但圆锥角膜中 II型胶原表达减低 ,在圆锥角膜基质斑块状瘢痕区 II型胶原呈强阳性表达。结论　 I、II型胶原的减少会导致角膜的稳定性降低 ,使角膜的机械抵抗力减弱 ,并使角膜前凸变薄。     

人工角膜植入术后EGF和bFGF局部应用的实验研究

目的 在兔眼局部应用上皮生长因子(EGF)及碱性成纤维细胞生长因子(bFGF)，观察对人工角膜植入术后植床角膜的影响。方法 于PMMA襻状支架人工角膜植入兔眼后，结膜下注射EGF或bFGF，术后1月取植床角膜制作石蜡切片，应用免疫组织化学技术ABC法，检测植床角膜组织增生细胞核抗原(PCNA)的表达情况及细胞凋亡情况；并检测c—myc基因的表达情况。结果 bFGF组植床角膜基质细胞可见PCNA阳性表达；EGF组植床角膜上皮细胞及基质细胞均可见PCNA阳性表达；对照组未见PCNA表达；各组植床角膜基质及上皮细胞均可见c—myc基因表达，角膜内皮细胞未见表达；各组均未见阳性凋亡细胞。结论 EGF和bFGF对人工角膜植入兔眼后植床角膜组织细胞的增生过程有促进作用，对减少人工角膜植入术后并发症，提高成功率有积极意义。     

人类转化生长因子诱导的细胞外基质黏附蛋白βig-h3在圆锥角膜和正常角膜中的表达

目的：观察圆锥角膜患者角膜组织及正常人角膜组织中人类转化生长因子诱导的细胞外基质黏附蛋白βig-h3的表达，探讨细胞外基质结构成分在圆锥角膜病变中的作用。方法：采用原位杂交技术，以BIGH3基因的cDNA探针对圆锥角膜患者的角膜组织及正常人角膜组织中βig-h3表达主要在基质层；圆锥角膜患者角膜基质层中βig-h3的表达随角膜病变的程度加深而减弱，严重者无βig-h3阳性表达。在圆锥角膜病变区与正常角膜交界处，βig-h3表达呈强阳性。结论：细胞外基质结构成分βig-h3在角膜纤维间质有序化的发育中起细胞黏附作用；圆锥角膜病变的发生过程可能与βig-h3的水平下降有关。     

Ⅲ型和Ⅳ型胶原在正常角膜和圆锥角膜中的表达

目的：观察正常人及圆锥有膜中胶原Ⅲ，Ⅳ型胶原的表达，探讨Ⅲ，Ⅳ型胶原在圆锥角膜病变中的变化。方法：采用免疫组织化学－SABC法检测角膜组织中Ⅲ，Ⅳ型胶原的表达。结果：Ⅲ型胶原在正常角膜和圆锥角膜的上皮屠，实质屠及内皮层均有阳性表达，但圆锥角膜比正常角膜的表达弱，而圆锥角膜基质斑块状瘢痕区可见Ⅲ型胶原呈强阳性表达；正常角和圆锥角膜组织中在Bowmans层，Descemets层和基质层均可检测到VI型胶原表达，但圆锥角膜中Ⅳ型胶原表达减低，特别在圆锥角膜的疤痕区内，Ⅳ型胶原明显降低，结论：Ⅲ，Ⅳ型胶原的异常表达会导致角膜基质织维间隙的稳定性降低，改变结缔组织中胶原束的组成，最终影响角膜的正常形态。     

圆锥角膜中细胞凋亡及Fas-L蛋白的表达

目的 探讨凋亡与圆锥角膜发病的关系及凋亡相关蛋白Fas-L的表达.方法 对20例圆锥角膜及5例正常角膜用原位末端标记法(TUNEL)检测凋亡,用免疫组织化学SP法检测Fas-L蛋白的表达;透射电镜观察凋亡细胞的形态学变化.结果 TUNEL染色示圆锥角膜组中上皮层、基质层及内皮层中细胞凋亡与正常角膜组比较差异均有统计学意义(P＜0.05);免疫组织化学示圆锥角膜组与正常角膜组基质层间Fas-L表达比较差异有统计学意义(P＜0.05);透射电镜可见圆锥角膜中存在凋亡特征的细胞.结论 圆锥角膜中存在凋亡,Fas-L蛋白的表达存在异常,Fas-FasL系统可能在圆锥角膜细胞凋亡中发挥了重要作用.     

大鼠角膜碱烧伤后碱性成纤维细胞生长因子在角膜中的表达及意义

目的 探讨大鼠角膜碱烧伤后成纤维细胞生长因子（bFGF）在角膜中的表达和意义。方法 采用碱烧伤大鼠角膜建立角膜炎症性新生血管动物模型；免疫印迹法检测大鼠角膜碱烧伤后不同时间段bFGF在角膜中的表达；免疫组织化学方法检测大鼠角膜碱烧伤后bFGF在角膜不同组织层次的表达。结果 免疫印迹法检测显示：去除上皮后的正常大鼠角膜无bFGF表达；碱烧伤后96h，大鼠角膜可检测出bFGF表达，随着时间进展，bFGF蛋白表达量逐渐增加。免疫组化结果：正常大鼠角膜上皮层表达bFGF；碱烧伤后角膜上皮层bFGF着染程度逐渐增强，在碱烧伤后48h以前，基质层浸润的炎症细胞无bFGF着染，96h以后，浸润的炎症细胞和新生血管内皮细胞均呈bFGF阳性。结论 bFGF未参与碱烧伤后角膜新生血管增殖的早期诱导，对角膜炎症性新生血管的增殖仅起到后续支持作用。     

正常人及圆锥角膜患者角膜中Ⅳ、Ⅴ型胶原的检测

目的：观察正常人及圆锥角膜患者角膜组织中Ⅳ、Ⅴ型胶原的表达，探讨基底膜损伤在圆锥角膜病变中的作用。方法：采用免疫组织化学SABC法检测角膜组织中Ⅳ、Ⅴ型胶原的表达。结果：正常角膜组织中Ⅳ型胶原表达在Bowman‘s层及Descemet‘s层，圆锥角膜患者Ⅳ型胶原表达也在Bowman‘s层及Descemet‘s层，但其阳性表达因角膜病变的程度不同而呈不同程度的减弱。Bowman‘s层的断裂呈波浪状，实质层排列不规则，局部可见瘢痕形成，Descemet‘s层膜破裂。角膜上皮，基质和内皮层未见明显的阳性表达，Ⅴ型胶原表达在Bowman‘s层及基质层，正常及圆锥角膜中无明显差异，但在圆锥角膜瘢痕区Ⅴ型胶原表达呈强阳性。结论：Ⅳ型胶原是基底膜的主要胶原，Bowman‘s层的断裂导致圆锥角膜的发病过程，最终整个过程以瘢痕形成结束。     

接合黏附分子-1在大鼠角膜中的表达研究

背景 接合黏附分子-1(JAM-1)是新发现的跨膜蛋白,参与细胞紧密连接的结构组成和功能发挥.在眼组织方面,紧密连接对维持角膜的透明性十分重要,但是目前就JAM-1在角膜紧密连接结构和功能方面的研究较少. 目的 确定JAM-1在大鼠角膜上皮层、基质层和内皮层的构成.方法 选取4只SPF级Wistar大鼠,2只用于JAM-1基因在角膜组织中表达的逆转录聚合酶链反应(RT-PCR)检测,另2只用于免疫组织化学检测.动物过量麻醉处死后获得角膜组织并制备角膜上皮、基质和内皮标本,RT-PCR法检测角膜标本中JAM-1、occludin和claudin-1 mRNA的表达.反应产物行质量分数1.5％琼脂糖凝胶电泳并用凝胶成像系统进行分析.用兔抗鼠JAM-1单克隆抗体对角膜石蜡切片、上皮及内皮铺片行免疫组织化学检测,评估JAM-1蛋白在大鼠角膜组织各层的表达部位和表达强度. 结果 在大鼠角膜各层均可检测到JAM-1、occludin和claudin-1 mRNA的表达,PCR熔解曲线为清晰的单峰.角膜组织各层中JAM-1 mRNA表达水平与occludin mRNA相似,均高于claudin-1 mRNA.3种黏附分子均在上皮层表达最强,角膜基质层表达较弱.免疫组织化学检测显示,JAM-1蛋白在角膜各层均有明确的阳性染色,角膜上皮基底层的表达强于基质层和内皮层.角膜上皮、内皮铺片检测显示,JAM-1蛋白主要表达于上皮细胞和内皮细胞的连接部位,而角膜内皮中JAM-1蛋白的阳性染色广泛而弥散.结论 JAM-1作为细胞连接的构成成分,在角膜上皮层、内皮层和基质层均有表达,但其表达的形态和水平因组织层次的不同而不同.     

白介素-18、血管内皮生长因子与角膜病变血管形成关系的研究

目的检测IL-18、VEGF在正常角膜和血管化角膜的表达,探讨IL-18在角膜新生血管化中的作用。方法收集角膜移植手术后病变角膜23例,正常角膜10例,采用免疫组织化学染色方法检测IL-18、VEGF在正常角膜和血管化角膜各层组织的表达。结果正常角膜组织中IL-18主要分布在上皮层、前弹力层、内皮层,以上皮层的表达最明显,VEGF在上皮层、前弹力层、和内皮层均有表达,前弹力层表达最强烈;血管化的角膜标本中IL-18表达相对正常角膜降低(P<0.001),而VEGF在该标本中上皮层、基质层均强烈表达,新生血管化区域、炎性细胞均呈现强阳性,相对正常角膜明显增强(P<0.001);二者表达呈负相关(γ=-0.683,P<0.05)。结论血管化角膜中IL-18水平明显降低,角膜新生血管化病理过程中新生血管的形成与IL-18水平降低密切相关。     

The Changes of TGF-α, TGF-β_1 and Basic FGF Messenger RNA Expression in Rabbit Cornea after Photorefractive Keratectomy

Objective : To study the mechanism of haze formation and investigate the expression changes of transforming growth factor-α(TGF-α), transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFCF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK).Methods: Sixteen white rabbits were randomly divided into 4 groups, and PRK was performed on each eye of 12 rabbits. The haze formation was examined under a slit-lamp microscope at the 1st, 2nd and 3rd month after PRK, and the expressions of TGF-α , TGF-β1 and bFGF mRNA were detected with in situ hybridization.Results : The corneal haze formed at the 1" month after PRK. The most prominent haze formation was observed at the 2nd month, and declined gradually at the 3rd month after ablation. TGF-a mRNA expression was presented on the normal corneal epithelium and not on the corneal stroma. TGF-β1 and bGFG mRNA were expressed by both corneal epithelium and stroma. The capacities for cornea tissue expression of thre     

The expression of protein betaig-h3 inducible by transforming growth factor-beta in keratoconus and normal cornea

OBJECTIVE: To observe the expression of a protein inducible by transforming growth factor-beta (betaig-h3) in normal cornea and keratoconus and discuss the effects of extracellular matrix on keratoconus. METHODS: In situ hybridization method was used to detect the expression of betaig-h3 in the cornea. The cDNA library was screened with human betaig-h3 cDNA probe to orient betaig-h3 mRNA in cells. The level of betaig-h3 was detected in normal cornea and keratoconus. RESULTS: betaig-h3 mainly expressed in the stroma in normal cornea and keratoconus. The positive expression decreased along with the increase of severity of the keratoconus. The negative expression was seen in severe lesions of keratoconus. The strong positive expression was seen at the interface area between the normal and the lesion of keratoconus. CONCLUSION: The decreasing of the level of betaig-h3 causes the diminution of corneal steadiness that is related to the formation of keratoconus.     

大鼠角膜化学伤后PEDF和VEGF的不平衡表达

目的比较硝酸银化学伤后大鼠角膜和正常角膜色素上皮衍生因子（PEDF）和血管内皮生长因子（VEGF）表达水平，揭示两者与角膜新生血管的相关性。方法10只大鼠左眼角膜硝酸银化学伤后为实验组，右眼为正常对照组，伤后15d行免疫组织化学法定位及Western blot定量检测样本角膜PEDF、VEGF等的表达。结果免疫组织化学检查：实验组角膜VEGF、碱性成纤维细胞生长因子（bFGF）强表达，PEDF未见表达或弱表达。正常组角膜PEDF高表达，VEGF弱表达，bFGF几乎不表达。Western Blot分析：实验组角膜PEDF表达明显下降（t=8．0049，P〈0．01），VEGF表达显著升高（t=48．3637，P〈0．01）。结论角膜严重化学伤后新生血管抑制因子PEDF破坏，刺激因子VEGF产生增加，PEDF／VEGF比值降低，角膜血管新生。     

Colocalization of fibroblast growth factor binding sites with extracellular matrix components in normal and keratoconus corneas

Basic and acidic fibroblast growth factor (bFGF, aFGF) binding sites were determined in frozen sections of normal and keratoconus corneas. After incubation with I-125 radiolabelled growth factors, corneal binding sites were revealed by autoradiography. The growth factors were localized mainly to Descemet's membrane and to the epithelial basement membrane. FGF binding sites were generally similar in normal and keratoconus corneas. The binding specificity was demonstrated by competitive inhibition experiments with an excess of unlabelled growth factors. The binding sites were sensitive to pretreatment of the corneal sections with heparitinase. We have attributed FGF's basement membrane affinity to one of its constituents, proteoheparan sulfate. Proteoheparan sulfate, laminin, collagen type IV, and fibronectin were all revealed by immunofluorescent techniques. While keratoconus cornea stroma had less laminin but more fibronectin than normal corneas the main difference lied in type IV collagen which was overexpressed in keratoconus epithelium.     

Immunohistochemical Expression of Fas Ligand (FasL) and Neprilysin (Neutral endopeptidase/CD10) in Keratoconus

Recent evidence demonstrated a correlation between apoptosis and neprilysin expression. The aim of this study was to investigate the immunohistochemical expression of Fas ligand (FasL) and neprilysin in keratoconic corneas in comparison to normal cadaver corneas to evaluate if such molecules play a role in the pathogenesis of keratoconus. We studied the expression of FasL and neprilysin in corneal specimens removed during penetrating keratoplasty in 15 cases with keratoconus and compared them with 5 normal cadaver corneas. In keratoconus, FasL was expressed in epithelium, endothelium and sub-Bowman’s stroma only, while neprilysin was expressed in epithelium, endothelium and all stromal layers. All normal corneas showed weak expression of both markers in basal epithelial layer only. In keratoconus, corneal epithelium with higher expression of FasL may evoke apoptosis in keratocytes, while neprilysin could prevent possible rescue of keratocytes from apoptosis.     

The expression of laminin-5 and ultrastructure of the interface between basal cells and underlying stroma in the keratoconus cornea

PURPOSE: We investigated the expression of laminin-5 and integrins, and the ultrastructure of the interface between basal cells and the basement membrane in the keratoconus cornea. These findings were compared to those in normal central cornea and limbus. METHODS: Frozen sections of the normal cornea (center and limbus) and the keratoconus cornea were immunostained with monoclonal antibodies against three chains of laminin-5 and integrins. To investigate the ultrastructure of the interface between basal cells and the underlying stroma, we used transmission electron microscopy. RESULTS: As compared to those in the normal central cornea, immunostaining patterns of the three chains of laminin-5 were thick and irregular in the keratoconus cornea and the normal limbus. Using electron microscopy analysis, the same characteristic structure of the interface between basal cells and the underlying stroma was recognized in the keratoconus cornea and the normal limbus. The expression of integrin alpha(6)beta(4) was restricted to the basal aspect of basal cells in the normal cornea. In the keratoconus cornea, however, integrin alpha(6)beta(4) was expressed in all aspects in basal and suprabasal cells. CONCLUSION The expression patterns of laminin-5 and the ultrastructure of the interface between basal cells and the basement membrane in the keratoconus cornea were similar to those in the normal limbus.     

The Changes of TGF—α,TGF—β1 and Basic FGF Messenger RNA Expression i

OBJECTIVE: To study the mechanism of haze formation and investigate the expression changes of transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta 1(TGF-beta 1) and basic fibroblast growth factor (bFGF) mRNA in corneal epithelium and stroma after photorefractive keratectomy (PRK). METHODS: Sixteen white rabbits were randomly divided into 4 groups, and PRK was performed on each eye of 12 rabbits. The haze formation was examined under a slit-lamp microscope at the 1st, 2nd and 3rd month after PRK, and the expressions of TGF-alpha, TGF-beta 1 and bFGF mRNA were detected with in situ hybridization. RESULTS: The corneal haze formed at the 1st month after PRK. The most prominent haze formation was observed at the 2nd month, and declined gradually at the 3rd month after ablation. TGF-alpha mRNA expression was presented on the normal corneal epithelium and not on the corneal stroma. TGF-beta 1 and bGFG mRNA were expressed by both corneal epithelium and stroma. The capacities for cornea tissue expression of three growth factors mRNA increased after PRK, and the peaks appeared on the 1st, 2nd month. The extent for expressions of three growth factors related proportionally to the haze formation. CONCLUSION: Three growth factors took part in promoting corneal wound healing after PRK, and might contribute to corneal haze formation and development.