Transplantation of reconstructed corneal layer composed of corneal epithelium and fibroblasts on a lyophilized amniotic membrane to severely alkali-burned cornea

The purpose of this article was to evaluate the graft efficacy of reconstructed corneal layer, composed of autologous corneal epithelium and fibroblasts on a lyophilized amniotic membrane (LAM), in a severely alkali-burned corneal model. After biopsy specimens were obtained from the left eyes of 24 rabbits, the corneal epithelial cells and fibroblasts were expanded in vitro and the corneal layer was reconstructed on LAM. Thirty-six eyes of rabbits underwent alkali burn (1 N NaOH, 30 s) to create a limbal deficiency and a deeply damaged corneal stroma. Four weeks later, group 1 underwent a graft of the reconstructed corneal layer composed of autologous corneal epithelium and fibroblasts on LAM. Group 2 was transplanted with a graft of the reconstructed autologous corneal epithelium, and group 3 served as a control without surgery. Wound healing and stabilization of the ocular surfaces occurred much faster in group 1 than in groups 2 and 3. The eyes in group 3 revealed typical limbal deficiencies with conjuctivalization and persistent corneal epithelial defects. However, the corneas in group 1 developed only mild peripheral neovascularization. Immunohistochemical staining in group 1 demonstrated that p63, cytokeratin 3, E-cadherin, transforming growth factor (TGF)-beta1, and collagen IV were expressed strongly in the corneal epithelium and basement membrane. On the basis of these results, transplantation of the reconstructed corneal layer, composed of autologous corneal epithelium and fibroblasts on LAM, partially accelerated the recovery of the alkali-injured rabbit ocular surface, and might be useful therapeutically for the treatment of patients with severely damaged cornea.     

人脐带血清器官培养液保存猪角膜的实验研究

目的 观察胎牛血清器官培养液和人脐带血清器官培养液对猪角膜内皮细胞形态学、组织学、超微结构、酶活性及代谢等的影响。方法 器官培养方法：4周以内31℃密闭培养,之后脱水24h。选择猪眼113只,其中100只角膜分为两组进行配对器官培养保存。第1组(50只角膜)：应用培养液Ⅰ(含10％胎牛血清);第2组(50只角膜,第1组的对侧角膜)：应用培养液Ⅱ(含10％人脐带血清)。13只角膜作为正常对照。器官培养每周每组各取出12只角膜进行内皮细胞形态学分析、HE染色、酶组织化学染色。保存2周、4周时行扫描电镜检查。检测保存前后培养液的pH值和葡萄糖、乳酸浓度。器官培养过程中行微生物学检测。结果 器官培养期间角膜内皮细胞单层保持完整,多形性增加,两组之间角膜内皮细胞密度、细胞面积的变异系数和六边形细胞比例在保存4周内差异无显著意义。保存4周的猪角膜与正常猪角膜相比,平均内皮细胞丢失率第1组为10．98％,第2组为10．85％。两组角膜器官培养后的组织学、超微结构、酶活性无明显区别。扫描电镜显示内皮细胞层完整,细胞形态改变。酶组织化学染色显示上皮、内皮细胞酶活性旺盛,基质细胞的酶活性随保存时间的延长而降低。角膜代谢状态良好。器官培养液污染率为6％。结论 两种器官培养液均可以保持角膜内皮活性达4周,推测人脐带血清能够替代胎牛血清作为角膜器官培养液的成分。(中华眼科杂志,2004,40：533-538)     

角膜中央厚度对压平眼压的影响及其临床意义

目的 :探讨高眼压症 (OHT)患者、原发性开角型青光眼 (POAG)患者与正常人角膜中央厚度 (CCT)的差别和CCT与压平眼压 (IOP)的关系 ,评价CCT测量对青光眼诊断的临床意义。方法 :以 131例、131眼为研究对象 ,包括 4 3例、4 3眼OHT ,30例、30眼POAG和正常人 4 8例、4 8眼。使用非接触眼压计测量IOP ,用超声角膜测厚仪盲法测量CCT。每例研究对象仅选 1眼用于统计学分析。结果 :正常组的平均CCT为 5 37.4±2 3.8μm ,性别之间无差异 (P =0 .978) ,CCT与年龄无关 (P =0 .0 82 )。回归分析显示平均CCT每增加 10 μm ,非接触眼压计测量值升高 0 .19mmHg。OHT组的CCT值为 5 90 .9± 2 0 .6 μm ,明显高于正常对照 (P =0 .0 0 1) ,POAG组的CCT为 5 15 .9± 36 .7μm ,则明显低于正常对照 (P =0 .0 0 1)。 结论 :OHT者的CCT高于正常人 ,而POAG患者的CCT低于正常人。CCT与压平IOP呈正相关。测量CCT对于正确评估压平眼压 ,判断患者是否存在损害视野的危险性及确定治疗的临界眼压具有重要意义。     

层粘连蛋白对兔角膜内皮细胞bcl-2表达的影响

目的：观察层粘连蛋白（LN）对兔角膜内皮细胞bcl-2表达的影响。 方法： 将体外培养的兔角膜内皮细胞分成3组：正常对照组、层粘连蛋白(LN)处理组、阴性对照组,采用免疫组化法和酶联免疫法分别检测3组的染色深浅及吸光度大小，以显示各组间bcl-2表达的水平。 结果： 免疫组织化学染色及评分结果显示LN组bcl-2为强阳性表达，正常对照组bcl-2为弱阳性表达，阴性对照组bcl-2为阴性表达。酶联免疫吸附试验检测3组吸光度分别为1.21±0.18（LN组）、1.05±0.14（正常对照组）和0.04±0.01（阴性对照组）。 结论： 层粘连蛋白能促进兔角膜内皮细胞bcl-2基因表达，从而可以有效地抗细胞凋亡。     

Corneal scar formation

The present study is concerned with scar formation in rabbit cornea after removal of a centrally-located 2·0 mm full thickness corneal “button”. Simplicity in wounding, reproducibility in healing with minimum complications, and ease of recovery of adequate amounts of “pure” scar tissue make this experimental model suitable for quantitative biochemical analyses.Quantification of hexosamine in purified glycosaminoglycan fraction from corneal excision wounds indicates that the amount of glycosaminoglycan progressively increases to 74% of that in the normal stroma by the fourth week of healing. This observation is not consistent with previous finding in corneal incision wounds.Hydroxyproline determinations in corneal wounds strongly suggest that collagen accumulation is very rapid during the first two weeks of healing. This is followed by a gradual increase in collagen, approaching the level in normal corneal stroma by the ninth week of healing.     

Corneal ulceration and the serum antiproteases. II. Complexes of corneal collagenases and alpha-macroglobulins.

Previous work has demonstrated that whole rabbit or human serum and the human α₂-macroglobulin (Hα₂-m) inhibit corneal collagenases. In the present study, the ability of rabbit and human serum to cause rabbit corneal collagenase, mol. wt 45 000, to elute in high molecular weight fractions from sieving columns is taken as presumptive evidence for the formation of collagenase-serum protein complexes. The recovery of increased collagenase activity by thiocyanate treatment of serum effluent fractions containing the human α₂-macroglobulin indicates that it is the α₂-m in human serum that complexes rabbit corneal collagenase. This conclusion is supported by the demonstration that purified Hα₂-m transports the rabbit corneal collagenase through a molecular sieve. Moreover, the chromatography of day one culture media from ulcerating rabbit corneas has demonstrated the presence of a significant amount of the total collagenase activity in the high molecular weight (850 000 – 1 000 000) fractions in which rabbit α-macroglobulin (Rα₁-m) was also demonstrated. These observations support the hypothesis that the α-macroglobulins play an important role in the regulation of corneal collagenase activity.Crossed-gel immunoelectrophoretic methods have shown that rabbit and human corneal collagenase preparations perturb the patterns of their respective α-macroglobulins. The perturbed patterns are taken as evidence for the formation of collagenase-α-macroglobulin complexes. The application of crossed-gel methods to the tears of human ulcer patients shows the utility of such methods for examining the status of α₂-m in tears.     

A new method to obtain epithelial and stromal explants from human Corneo-Scleral Discs for the routine culture of corneal epithelial and fibroblast cells

Acquisition of human corneal cells for culture is hindered not only by the scarcity of donor tissues but also by some of the standard enzymatic and mechanical isolation techniques. Good yields have been reported from full-thickness explant and sclero-limbal pieces. However, due to their greater proliferative capacity, fibroblasts will encroach and subsequently overwhelm epithelial cultures whichever technique is used. The novel approach presented here is to minimise this by removal of the whole stroma from the epithelial layers at the outset. This is achieved by selective sectioning with the Webb mini-microtome developed in the Norwich Eye Research Laboratory. The microtome can be sterilised by alcohol spraying or autoclaving and is small enough to use in the culture hood. A selective cut in the region of the Bowmans membrane results in the isolation of the epithelium from the stroma and thus exposed, the basal epithelial layers are released from contact inhibition to allow growth. The stroma is further cut to produce multiple sections for the culture of fibroblasts. Both pure epithelial and stromal fibroblast cultures have been successfully generated in serum-enriched medium as well as defined serum-free media with growth supplements, from the corneo-scleral discs of donors of all ages.     

近视眼角膜屈光力测定分析

目的：探讨近视眼角膜屈光力与屈光度的关系。方法：采用计算机辅助的角膜地形图仪（ＴＭＳ－２）测定角膜中央６～８环内的最大子午线屈光力（ＳｉｍＫ１）及与其呈９０°子午线的屈光力（ＳｉｍＫ２）。结果：近视眼角膜屈光力与总体屈光度之间呈极显著负相关性，低、中、高度及超高度近视组之间ＳｉｍＫ１、ＳｉｍＫ２差异无显著性。结论：近视眼屈光度低与角膜屈光力相关性大，角膜屈光力在不同屈光度之间无显著性差异     

穿透性角膜移植术治疗假晶体大泡性角膜病变的临床观察

目的：评价穿透性角膜移植术治疗假晶体大泡性角膜病变的临床疗效，探讨术中对原人工晶体的处理。方法：假晶体大泡性角膜病变１４例（１４只眼）。原人工晶体类型为后房型人工晶体１２例，弹性开放襻前房型人工晶体２例。手术方法为穿透性角膜移植术１２例，穿透性角膜移植联合人工晶体取出术２例。结果：术后临床症状均完全消失，所有植片均透明，矫正视力０．０２以上１２例（０．１以上４例）。随诊３￣２２个月，１３例植片透明     

准分子激光原位角膜磨镶术治疗近视的角膜地形图分析

应用角膜地形图仪对２１例（３０只眼）近视患者于准分子激光原位角膜磨镶术（ｌａｓｅｒｉｎｓｉｔｕｋｅｒａｔｏｍｉｌｅｕｓｉｓ，ＬＡＳＩＫ）后进行角膜地形图的检测分析，发现ＬＡＳＩＫ术后角膜表面的球面形状发生一定的改变：ＳＲＩ和ＳＡＩ与术前比较均有显著性差异。术后３个月检查时，激光切削区偏心０．５６±０．３５ｍｍ；角膜地形图主要表现为４种形态：圆形或椭圆形（４６．７％）、哑铃形（１０．０％）、半环形（６．７％）、中央小岛形（３６．７％），且各种形态与最佳视力有着一定的关系。由此证明，角膜地形图的定量分析对ＬＡＳＩＫ术后角膜表面球面形状的评价、手术设计的改进、手术疗效的预测等是有重要的临床意义的