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。     

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。     

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。     

正常角膜与圆锥角膜中的I型、VI型胶原与金属蛋白酶—2

目的：对比正常角膜与圆锥角膜中的I型，VI型胶原及金属蛋白酶－2的定位与含量。方法：应用酶免疫组化及间接免疫荧光技术检测正常与圆锥角膜中I型、VI型胶原及金属蛋白酶－2（MMP－2）的定位。阳性结果应用图像分析系统进行定量分析。结果：免疫组化结果显示所有角膜中I型胶原存在于弹力层和基质中；VI型胶原的含量无差别MMP－2的含量明显增多，差异有显著性。结论：圆锥角膜的MMP－2含量增多，其所导致的胶原降解异常可能是圆锥角膜发病的关键。     

正常角膜与圆锥角膜中的Ⅰ型、Ⅵ型胶原与金属蛋白酶-2

目的对比正常角膜与圆锥角膜中的Ⅰ型,Ⅵ型胶原及金属蛋白酶-2的定位与含量。方法应用酶免疫组化及间接免疫荧光技术检测正常与圆锥角膜中Ⅰ型、Ⅵ型胶原及金属蛋白酶-2（MMP-2)的定位。阳性结果应用图像分析系统进行定量分析。结果免疫组化结果显示所有角膜中Ⅰ型胶原存在于前弹力层和基质中;Ⅵ型胶原存在于上皮基底膜、前弹力层、基质、后弹力层;MMP-2存在于上皮、基质、内皮中。同正常角膜比,圆锥角膜中Ⅰ型、Ⅵ型胶原的含量无差别而MMP-2的含量明显增多,差异有显著性。结论圆锥角膜的MMP-2含量增多,其所导致的胶原降解异常可能是圆锥角膜发病的关键。     

圆锥角膜组织中EGF和bFGF变化免疫组织化学的研究

目的 观察正常人及圆锥角膜中表皮生长因子(EGF)和碱性成纤维细胞生长因子（bFGF)的表达，探讨EGF和hFGF在圆锥角膜中的变化。方法 采用免疫组织化学SABC法检测角膜组织中EGF、bFGF的表达，结果 在圆锥角膜上皮层、Bowman层和内皮层有EGF表达，EGF比正常角膜的表达强；在圆锥角膜的上皮层、实质层和内皮层可见bFGF的阳性表达，圆锥角膜的基质瘢痕区bFGF有较强的阳性表达。结论 圆锥角膜组织中EGF水平增高，促进上皮细胞和基质细胞的DNA合成，从而使组织愈合。bFGF表达增强可以促进角膜上皮、基质细胞和内皮细胞的增殖。     

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。     

圆锥角膜的免疫组织化学研究

目的：了解圆锥角膜基底膜免疫组化的变化,以发现圆锥角膜可能的特异性改变,为探讨其发病机制提供依据。方法：收集圆锥角膜在我院眼科行穿透性角膜移植术的角膜片,用链霉亲和素－生物素法（SP法）检测圆锥角膜、角膜白斑、正常角膜基底膜Ⅳ型胶原、Fn、Ln表达的异同,并进行统计学分析。结果：圆锥角膜基底膜Ⅳ型胶原,Fn染色较正常角膜显著升高,但与角膜白斑比较无显著性差异。结论：圆锥角膜基底膜的改变可能与创伤愈合有关。提示圆锥角膜原发病变可能在角膜基质细胞。眼科学报2000;17：65－67。     

大鼠角膜新生血管生长与其周围基质内相关生物因子表达关系的研究

目的 探讨角膜新生血管周围间质相关生物因子与新生血管生长之间的关系.方法 实验研究.40只Wister大鼠采用NaOH滤纸烧灼法制作碱烧伤角膜新生血管模型.术后1、3及7 d,免疫荧光法检测角膜新生血管周围角膜基质间质相关生物因子包括转化生长因子β1(TGF-β1)、α平滑肌肌动蛋白(α-SMA)、成纤维细胞激活蛋白(FAP),并观察它们与新生血管之间的关系,以血小板内皮细胞黏附因子(CD31)标记血管内皮细胞.采用RT-PCR方法 检测术后3、7 d的FAP在角膜中不同位置的表达.苦味酸天狼猩红-偏振光法检测术后7 d角膜基质中主要胶原Ⅰ型和Ⅲ型胶原的改变.结果 碱烧伤后1、3及7 d角膜冰冻切片免疫荧光单染与双染显示,角膜基质首先出现TGF-β1阳性表达,然后随着新生血管的形成出现α-SMA、FAP同时阳性的细胞,正常角膜组织内无阳性表达.FAP阳性基质细胞位于CD31阳性的血管内皮细胞周围,与血管内皮细胞伴行生长,两者无明显的先后顺序.RT-PCR结果 显示,新生血管生长到达的位置出现FAP阳性表达.角膜新生血管生长后基质中Ⅰ型、Ⅲ型胶原重新排列.结论 角膜新生血管形成时,血管周围基质相关生物因子发生了改变,出现了FAP阳性的基质细胞,此细胞包绕并伴随血管内皮细胞生长;角膜基质中Ⅰ型、Ⅲ型胶原蕈新排列以适应新生血管的生长.(中华眼科杂志,2009,45:158-163)     

准分子激光术后大鼠角膜上皮下雾状混浊中Ⅰ型前胶原的测定

目的探讨准分子激光术后大鼠角膜中Ⅰ型前胶原的变化,比较角膜非手术区、手术野透明区与上皮下雾状混浊（haze）区的Ⅰ型前胶原含量的差异。方法32只6月龄清洁级SD大鼠按照手术前后被处死的时间不同平均分为8组。28只大鼠的一侧眼被施行准分子激光角膜切削术（PRK）。术后每日裂隙灯显微镜下观察术眼角膜情况,角膜haze的分级参照Seiler标准。分别于手术前和术后1、3、7、15d及1、2、3个月处死动物,行苏木精-伊红染色,观察手术区角膜的形态变化,采用免疫组织化学法检测角膜Ⅰ型前胶原在术眼角膜组织中的表达,并以光密度（OD）值表示。对不同时间采集的角膜非手术区、手术野透明区与haze区中Ⅰ型前胶原含量进行定量分析。结果臼术后3d起,术眼角膜上皮开始移行覆盖切削区并逐渐增厚,基质纤维细胞增生活跃;术后1个月时术眼角膜上皮基底膜开始形成,基质胶原纤维排列紊乱;术后3个月角膜基质胶原重塑过程基本完成,角膜结构趋于正常。大鼠角膜术后haze分级与Ⅰ型前胶原含量呈正相关（r=0．406,P〈0．05）;非手术区各时间段角膜平均OD值均无明显变化（t=5．719,P〉0．05）;手术区角膜中的Ⅰ型前胶原含量明显高于非手术区（P〈0．05）;haze区平均OD值明显高于非haze区（t=5．578,P〈0．05）。免疫组织化学检测表明,角膜上皮中Ⅰ型前胶原的阳性表达在术后7d最多,并终止于术后1个月,而角膜细胞间质中Ⅰ型前胶原持续至整个修复过程。结论Ⅰ型前胶原含量的增加可能是haze形成的主要因素之一,为进一步研究准分子激光术后角膜前胶原的变化提供理论支持。     

Biochemical investigation of cells from keratoconus and normal cornea

Collagen is the major structural protein in the cornea. In keratoconus the central cornea is thin, opaque and weak. Collagen synthesis was investigated in cells derived from the stroma of cornea with keratoconus and from controls. No difference was found in the ratio of collagens type I and type III synthesized, which were investigated as procollagens and after conversion to collagen as well. The $\text{α1}\text{α2}$ ratio in type I collagen was similar in keratoconus cells and controls.     

Distribution of types I, II, III, IV and V collagen in normal and keratoconus corneas

By using type-specific antibodies to types I, II, III, IV and V collagens, distribution of distinct types of collagen in normal human cornea as well as keratoconus cornea were examined by indirect immunofluorescence microscopy. In normal human cornea, immunohistochemical evidence supported the previous biochemical finding that type I collagen was the major type of collagen in human corneal stroma. No reaction was observed to anti-type II collagen antibody in the whole cornea. Anti-type III collagen antibody reacted with the corneal stroma in a similar fashion as that of anti-type I collagen antibody. Type IV collagen was observed in the basement membrane of the corneal epithelium and in Descemet's membrane. Anti-type V collagen antibody also reacted with the corneal stroma diffusely. Bowman's membrane was strongly stained only with he anti-type V collagen antibody. For further details of the distribution of type I, type III and V collagens in human corneal stroma, immunoelectron microscopic study was undertaken. The positive reaction products of anti-type I and anti-type III collagen antibodies were located on the collagen fibrils, while that of anti-type V collagen antibody was either on or close to collagen fibrils. In keratoconus cornea, no difference was observed in terms of the distribution of type I, III and V collagens, while the disruptive and excrescent distribution of type IV collagen was noted in the basement membrane of the corneal epithelium.     

A comparative study of elasmobranch corneal and scleral collagens

Corneas, dissected from young and adult spiny dogfish sharks (Squalus acanthias), were prepared for transmission electron microscopy and immunofluorescence. In the latter case, tissues were fixed in formaldehyde solutions, sectioned with a cryostat, incubated with antibodies specific for collagen types I and II, and examined by indirect immunofluorescence. Collagen α- and β- chains were separated by sodium dodecylsulfate-slab gel electrophoresis and characterized by two-dimensional mapping of ¹²⁵I-labeled peptides generated by tryptic and chymotryptic digestion.The corneal stroma, the sutural fibers which span the stroma, and the surrounding limbus were positive for type I collagen, as judged by immunofluorescence. The corneal stroma was negative for type II collagen. Scleral cartilage matrix was intensely positive for type II collagen, but was negative for type I. In addition, the perichondrium of the scleral cartilage was positive for type I collagen. In confirmation of these results, slab gel electrophoresis revealed α1, and α2-like bands from shark corneal stroma, but only an α1-like band from shark cartilage collagen. Two-dimensional peptide mapping revealed some degree of resemblance between the α1 band of shark corneal stroma and the α1 band of chick type I collagen. Likewise, the α1 band of shark cartilage collagen somewhat resembled the α1 band of chick type II collagen. The α2-like band of shark corneal stroma did not closely resemble the α2 band of chick type I collagen. The most prominent β band of shark corneal stroma appeared to be a dimer composed of one α1 chain and one α2 chain. The collagen of shark corneal stroma was very susceptible to degradation by pepsin, whereas that from shark cartilage was much less susceptible.     

Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.     

Connective tissue changes associated with inferior epiblepharon, epicanthus, and keratoconus

A 34-year-old woman with bilateral inferior epiblepharon, epicanthus, and keratoconus is presented. Biopsies of right upper arm skin, left lower eyelid, and left cornea were sectioned and examined by electron and indirect immunofluorescent microscopy. Pathologic changes in both the skin and cornea were observed. Indirect immunofluorescent techniques showed an increase in fibronectin and procollagen type I in the subcutaneous tissue of the skin and an increase of procollagen type I in the deep dermis of the right upper arm skin and left lower eyelid. Increased amounts of laminin in the basal epithelium of the cornea and of collagen type III in the stroma and subepithelial components of the stroma were observed. Electron microscopy disclosed disordered arrays of collagen fibers in the right upper arm skin and left lower eyelid, and typical changes of keratoconus, with scars in the cornea. Both the cornea and skin had thin fibrillar extensions between collagen fibers.     

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.     

Electron microscopic and immunohistochemical examination of scarred human cornea re-treated by excimer laser

PURPOSE: To elucidate differences, at the macromolecular level, in corneal tissue subjected to repeated argon fluoride excimer treatment. METHODS: A light microscopic, electron microscopic, and immunohistochemical study was performed on a scarred human cornea. RESULTS: Keratocytes were enlarged with an expanded endoplasmic reticulum and exhibited a fibroblastic appearance. Amorphous material was observed extracellularly. Collagen fibrils exhibited a disordered arrangement while banding patterns and diameter were normal. Immunohistochemical investigation of several collagen types, of collagen-associated proteoglycans, and of basement membrane components demonstrated an enhanced immunoreactivity of all of them in the scarred area. Type V collagen was found as a normal component of the epithelial basement membrane whereas types I and III collagen were present beneath Bowman's layer. Excimer-laser-treated sections revealed considerably stronger subepithelial staining for collagen types I, III, IV, and V. Laminin-1, a typical component of basement membranes, was detectable throughout the scarred tissue. The small proteoglycans decorin and fibromodulin accumulated in a patch-like manner in the scarred tissue below the epithelium, whereas biglycan was expressed by the epithelium and throughout the stroma. Lumican was expressed most strongly by the epithelium and rather equally distributed in the excimer-laser-treated and in the normal stroma. CONCLUSION: Effects of argon laser treatment of the cornea must be regarded as a process acting over many months. Intra- and extracellular structures and components are involved and influence the unpredictable shape of the corneal architecture.     

Discoidin domain receptor (DDR) 1 and 2: collagen-activated tyrosine kinase receptors in the cornea

Discoidin domain receptor (DDR) 1 and 2 have recently been found to serve as receptors for several collagen types. These receptors have been found to modulate cell proliferation and metalloprotease expression in response to collagen stimulation. The purpose of this study was to examine expression of DDR1 and DDR2 in the cornea and to determine the effect of several collagen types on proliferation and response to pro-apoptotic cytokines by corneal fibroblasts. DDR1 and DDR2 mRNAs were detected by RT-PCR. Proteins were detected by immunocytochemistry and immunoprecipitation with Western blotting. Cell proliferation in response to acetic acid-solubilized collagen type I, II, IV, IX or X was determined by cell counting. The effect of these collagen types on Fas-stimulating antibody-induced cell death was determined by trypan blue assay. DDR1 and DDR2 mRNAs were detected in each major human cell type of the cornea. Both were also detected in ex vivo human corneal epithelium. DDR1 and DDR2 proteins were detected in all three major cell types in culture and in human corneal tissue. Collagen types I, II, IV, IX and X stimulated proliferation, but had no effect on Fas-mediated apoptosis, of corneal fibroblasts. DDR1 and DDR2 tyrosine kinase receptors are expressed in the cornea. Collagen-stimulated mitosis of corneal fibroblasts in culture is likely mediated by the DDR receptors. Collagen had no effect on Fas-mediated apoptosis of corneal fibroblasts.