以异种后弹力膜基质为载体培养角膜内皮细胞的研究

目的　探讨以异种后弹力膜 (DM) /基质为载体培养角膜内皮细胞的可行性 ,为培养内皮细胞的临床移植提供新的方法和供体材料。方法　取厚 10 0 μm的猪角膜后层 ,冻存解冻后置中性蛋白酶 3 7℃孵育 5min ,去除内皮细胞 ,保留DM及薄层基质 ,置纯甘油中脱水保存 6个月 ;使用前复水 ,紫外线二面灭菌 ,制备成载体 ;接种兔角膜内皮细胞于载体的DM面并体外培养 ,行细胞形态、密度、组织学、超微结构的观察。结果　兔内皮细胞在猪DM/基质载体上贴附生长 ,形成紧密连接的单层 ,形态近似六角形分布 ;细胞密度达 ( 3 62 0± 110 2 3 )个 /mm2 ,具有正常兔角膜内皮细胞的超微结构。结论　角膜内皮细胞能够在异种DM/基质载体上良好生长 ,该载体有可能成为培养内皮细胞应用于临床移植的供体材料     

以羊膜为载体培养角膜内皮细胞的实验研究

目的 以羊膜为载体培养角膜内皮细胞并形成单层组织，为以后内皮细胞层移植提供方法和材料来源。方法 胰酶消化去除羊膜上皮细胞，保留其底膜作为载体，种植兔角膜内皮细胞，体外培养形成单层内皮层，并进行形态，组织学，超微结构观察。结果 角膜内皮细胞在羊膜上黏附生长并增殖，体外培养6-7d即融合成单层，其形态及生长密度优于单纯培养皿上培养的内皮细胞。复合角膜内皮组织由羊膜基底膜和单层扁平内皮细胞组成，与生理状态下的角膜内皮组织相近。结论 以羊膜为载体培养角膜内皮细胞可形成单层细胞层，有望成为角膜内皮移植良好的材料来源。     

以干燥脱水法保存的异种角膜基质为载体构建人工生物角膜上皮组织

目的:观察以干燥脱水法保存的鸵鸟角膜基质为载体构建人工生物角膜上皮组织的生物学特性。方法:采用组织块培养法获得新西兰大白兔角膜缘干细胞,经胰蛋白酶消化法获得细胞,种植于干燥脱水法保存的鸵鸟角膜板层基质上,采用气液界面培养法进行培养,通过倒置显微镜、透射电子显微镜、荧光显微镜观察其形态学、生长特点,超微结构及免疫学特征。结果:在干燥脱水法保存的鸵鸟角膜基质上种植兔角膜缘干细胞,接种72h后,细胞形成单层,移置气液交界面后继续培养7～10d,逐渐形成复层。经光镜、透射电镜、及免疫学检测显示其具有角膜上皮组织的生物学特性。结论:兔角膜缘干细胞能够在干燥脱水法保存的鸵鸟角膜基质载体上生长,并可形成复层,基本具有正常角膜上皮细胞的形态、超微结构和生物学特性。     

羊膜载体培养标记兔角膜内皮细胞移植的研究

目的探讨以羊膜基底膜为载体培养兔角膜内皮细胞移植的可能性,为培养内皮细胞移植治疗角膜内皮失代偿疾病提供依据与方法。方法体外培养扩增兔角膜内皮细胞,采用亲脂性碳青染料(CM-DiI)对细胞进行标记,种植于去除上皮细胞的羊膜基底膜上,体外培养形成单层角膜内皮层,并进行形态学、组织学、超微结构及细胞标记情况的观察;将羊膜为载体培养的内皮层对切除后板层的兔角膜进行移植,同时以无培养内皮细胞的羊膜移植和单纯角膜后板层切除为对照,术后观察角膜透明度与厚度,对其进行组织学与细胞标记情况的检测。结果 5～7d 角膜内皮细胞即在羊膜基底膜上融合成单层,细胞为扁平多角形,排列紧密,密度可达(3202.84±347.77)个/mm~2,荧光显微镜下可见内皮细胞被 CM-DiI 标记后显现红色荧光;培养内皮层移植后角膜维持相对的透明与薄度,而无内皮细胞羊膜移植和单纯后板层切除两组对照角膜严重水肿、混浊,厚度明显超过实验组角膜;培养内皮移植后角膜形成新的内皮层,通过标记的细胞发现移植后细胞仍为培养移植的内皮细胞而非周边细胞的移行。结论羊膜基底膜是角膜内皮细胞生长和移植的良好载体,体外培养角膜内皮细胞移植有望代替供体角膜移植,具有广阔的应用前景。(中华眼科杂志,2006,42:925-929)     

猪脱细胞角膜基质组织结构特点与生物相容性研究

背景构建组织工程化角膜时,载体的选择十分重要。目前有多种载体可供选用,但脱细胞角膜基质是公认的较好载体。目的观察猪脱细胞角膜基质的组织结构特点,评价其与异种角膜基质和上皮细胞的生物相容性。方法取猪角膜组织进行组织块培养,经胰蛋白酶-EDTA酶消化角膜上皮、基质、内皮细胞,支架组织于-20℃冷冻干燥后灭菌保存。猪脱细胞角膜基质石蜡切片行常规苏木精一伊红染色,光学显微镜下观察组织的形态学特征;扫描电镜下观察其组织结构特点;并对其物理特性,如抗拉性、膨胀性、含水量和透明度进行评价。将猪脱细胞角膜基质移植到兔角膜基质层内,同时与体外培养的兔角膜上皮细胞共培养4周,分析其组织和细胞生物相容性。结果经酶消化处理后猪角膜组织中未见上皮、基质和内皮细胞,其胶原纤维直径大小、排列与正常角膜组织相同,抗拉性、膨胀性、含水量和透明度等物理特性与正常猪角膜相似。猪脱细胞角膜基质行异种兔角膜基质层间移植1周时可见轻度水肿,2周后水肿基本消退,4周时透明度较好。猪脱细胞角膜基质与兔角膜基质之间贴附良好,未见炎症反应及新生血管。兔角膜上皮细胞接种于猪脱细胞角膜基质上共培养4周后CK3表达阳性。猪脱细胞角膜基质脱水前与脱水2h、4h后及正常猪角膜基质间的抗拉性、膨胀性、含水量的差异均无统计学意义（P〉0．05）,但脱水2h、4h后及正常猪角膜基质的透明度明显好于脱水前,差异均有统计学意义（P〈0．01）。结论猪脱细胞角膜基质组织结构与正常猪角膜相似,与兔角膜基质和上皮细胞具有良好的生物相容性。     

角膜内皮细胞改良培养技术的初步探讨

目的 评价角膜后弹力层撕除联合酶消化法分离角膜内皮细胞的效率,分析细胞体外生长的生物学特性.方法 将兔角膜带有内皮细胞的后弹力层完整撕下,用胰蛋白酶-EDTA联合酶消化,纯化的角膜内皮细胞进行体外培养.观察细胞形态,用神经元烯醇化酶抗体进行细胞表型鉴定.苏木精-伊红染色以及茜素红-台盼蓝联合染色分析细胞活性,流式细胞仪检测细胞体外生长过程中Anne xiv-PE的表达情况,透射电镜和扫描电镜进行细胞超微结构形态的观察.结果 带角膜内皮细胞的后弹力层撕除联合酶消化法可快速获得大量纯化的角膜内皮细胞,快速贴壁生长并增生,体外培养3～4d即融合成单层细胞,且表达神经元烯醇化酶抗体阳性,苏木精-伊红染色及活性染色提示细胞功能良好,Annexiv-PE抗体表达水平微弱.结论 角膜后弹力层撕除联合酶消化法可提高角膜内皮细胞的获取和培养效率,为工程化角膜内皮移植膜的构建提供稳定的种子细胞来源.     

体外培养角膜内皮细胞移植的实验研究

目的　探讨体外培养角膜内皮细胞移植的手术方法及移植后在活体的生理功能。方法　植片 :以直径 7 5mm、厚 10 0 μm、冷冻脱水保存的猪角膜后弹力膜 (Descemet′smembrane ,DM ) /基质为载体 ,原代接种兔角膜内皮细胞 ,体外培养 7～ 10d ,用于移植。受眼 :新西兰兔 3 0只 ( 3 0眼 ) ,实验组 2 0眼 ,对照组 10眼 ,全角膜范围去除术眼内皮细胞 ,5周后施行手术 ;做直径 9mm、3 / 4角膜厚的带蒂前层角膜瓣并掀置一侧 ,再用直径 7 2 5mm的环钻钻去中央后层角膜 ,形成植床 ;将植片置于植床 ,8-0可吸收线间断缝合 ,10 -0尼龙线间断缝合前层角膜瓣 ,术后观察 1～ 12个月。结果　术后角膜持续透明 6个月 14眼 ( 14 / 18、 77% ) ,12个月 8眼 ( 8/ 14、 5 7% ) ;术后 6个月内皮细胞密度 ( 193 4± 3 0 7)个 /mm2 ,角膜厚度平均为 ( 3 92± 3 9) μm。 结论　以异种DM /基质为载体培养的兔角膜内皮细胞 ,行同种异体移植后 ,能够在活体上成活 ,并具有正常内皮细胞的生物学功能 ,维持角膜透明 ,深板层角膜内皮移植术是体外培养内皮细胞移植的一种有效术式。     

体外重建组织工程人角膜内皮在新西兰兔角膜内皮移植中的应用

目的:验证体外重建组织工程人角膜内皮(TE-HCE)在角膜内皮移植中的作用。方法:以非转染人角膜内皮细胞(HCE细胞)为种子细胞,以去上皮层修饰羊膜为载体支架体外重建的TE-HCE,经CM-DiI标记后对撕除内皮层和后弹力层(DM)的新西兰兔进行了兔穿透性角膜内皮移植。用裂隙灯显微镜观察移植眼角膜的透明度。用荧光显微镜观察种子细胞荧光标记。用茜素红染色和冰冻切片HE染色和扫描电镜观察种子细胞的形态、细胞连接的形成情况、细胞单层的完整性及其与DM结合的紧密程度。用透射电镜方法鉴定种子细胞、DM和角膜的超微结构。结果:TE-HCE可使新西兰兔角膜保持透明39d以上。角膜内皮层移植区的细胞均带有CM-DiI荧光标记。绝大多数种子细胞为六角形,细胞间连接紧密,重建出了完整的角膜内皮层,且内皮层与DM结合紧密。种子细胞重建出了连续的角膜内皮层,种子细胞、DM和角膜的形态结构与正常对照眼的几乎相同。结论:移植后的TE-HCE在种子细胞形态、连续单层状态、细胞连接形成及超微结构上均与对照兔眼的角膜内皮类似,使移植新西兰兔角膜长期保持透明。     

猴,兔角膜细胞成分原代培养的研究

目的 建立从同一角膜上上分离培养角膜上皮,基质和内皮细胞的方法。方法 采用消化法培养猴角膜内皮细胞,上皮细胞和兔角膜上皮细胞,组织块培养法培养角膜基质细胞和兔角膜内皮细胞,结果 猴,兔角膜内皮细胞培养１周后,均能形成细胞单层,猴角膜上皮细胞培养３－４天生长旺盛,但难以形成细胞单层,兔上皮细胞生长旺盛,１周后已达融合状态,细胞呈膜状伸出板层伪足,猴,兔基质细胞易培养,１周后融合成单层细胞,结论 从同     

组织工程角膜内皮移植研究进展

由于角膜供体材料严重短缺, 穿透角膜移植术及角膜内皮移植术的临床广泛开展受到严重制约, 其根本原因在于健康角膜内皮的增生能力有限。随着组织工程技术和细胞工程技术不断发展, 组织工程角膜研究已取得一定进展, 应用组织工程技术体外培养高密度、具备健康内皮功能的角膜内皮细胞进行移植是当前研究的热点。组织工程角膜内皮技术研发的关键在于种子细胞、载体材料和移植方式的选择。目前, 国内外大量研究的种子细胞来源包括人角膜内皮细胞、干细胞、血管内皮细胞及人羊膜上皮细胞等。常见的载体材料包括羊膜、脱细胞角膜基质、后弹力层、晶状体前囊膜等。体外培养的细胞采用穿透角膜移植术、角膜内皮移植术或前房注射细胞的方式进行移植。本文从角膜内皮种子细胞来源、移植载体选择以及角膜内皮移植方法等方面就组织工程角膜内皮移植研究进展进行综述, 总结目前研究面临的问题并展望其前景。     

Relationship Between Corneal Hysteresis and Corneal Resistance Factor with Other Ocular Parameters

Abstract

Purpose: To evaluate the relationship between corneal hysteresis (CH) and corneal resistance factor (CRF) with age, central corneal thickness (CCT), corneal curvature (KM), corneal volume (CV), and refractive error in naïve eyes. Methods: 105 healthy subjects (58 male and 47 female) were included in this study. The ages ranged from 19 to 82 years (mean 43.1?±?15.4 years) and refraction between ?11?D and +6?D (mean ?0.79?±?2.95?D). CH and CRF obtained with the Ocular Response Analyzer (ORA) were correlated with age, refractive error, Goldmann Applanation Tonometry (GAT), and with CCT, KM, CV obtained with the Pentacam, and with Corneal-Compensated Intraocular Pressure (IOPcc) and Goldmann-correlated intraocular pressure measurement (IOPg) obtained with ORA. A multivariable mixed effect model was used to evaluate associations among these parameters. Results: CH ranged from 6.9 to 14.6?mmHg (mean 10.26?±?1.49?mmHg); CRF ranged from 5.8 to 17?mmHg (mean 10.38?±?1.64?mmHg). Multivariate analysis showed a statistically significant correlation between CH with CCT (p?<?0.001), and KM (p?<?0.001), and between CRF with CCT (p?<?0.001) and GAT (p?<?0.001). Conclusions: Our findings support the hypothesis that CH and CRF are related to the corneal shape and thickness, and show a decrease of CH with age.     

A case of congenital corneal anesthesia with hypopyon in china

 PURPOSE: To report a case of congenital corneal anesthesia (CCA) associated with hypopyon and tactile hypoesthesia. METHODS: Case report. RESULTS: A 3-year-old girl presented with redness and mild photophobia in the left eye accompanied by corneal ulcer, hypopyon, and corneal neovascularization. Corneal sensation was reduced bilaterally. She exhibited an absence of normal response to painful stimuli. She also had extensive ulceration of the lateral borders and tip of the tongue. The left corneal biopsy revealed negative cultures for bacteria and fungi. She was treated with human amniotic membrane transplantation in the left eye. The response to treatment was good. CONCLUSION: This is the first case of CCA reported in China. CCA is often misdiagnosed as infectious keratitis. Amniotic membrane transplantation is effective in repairing the severe corneal ulcer which may be associated with CCA.     

Biomechanics and structure of the cornea: implications and association with corneal disorders

Recent studies have shown that alterations in corneal biomechanical properties are associated with corneal pathologies, particularly corneal ectasia. Moreover, these alterations may have implications with regard to the outcomes of therapeutic modalities and corneal refractive surgeries. We address corneal anatomy and its relevance to corneal biomechanical characteristics, as well as ocular and systemic conditions associated with changes in corneal biomechanics.     

A new multiple noncontinuous puncture (pointage) technique for corneal tattooing

AIM:To assess the safety and cosmetic efficacy of a new multiple noncontinuous transepithelial puncture technique for tattooing a decompensated cornea.METHODS:It was anon-comparative clinical case series study.The study examines 33 eyes in 33 patients with total corneal opacity due to corneal decompensation, which developed following intraocular surgery.Corneal tattooing was performed using the multiple noncontinuous transepithelial puncture technique (i.e. pointage). The safety of this new surgical strategy was assessed by occurrence of adverse events for the follow-up period. The cosmetic efficacy was determined by the patient’s cosmetic satisfaction and independent observer’s opinion about patient appearance.RESULTS:Seven women and 26 men were included in the study. The mean age was 46.4±17.5y (range:7-67). In total, 30 of 33 patients (91%) reported cosmetic satisfaction within the follow-up period. Only 3 patients (9%) required additional tattooing due to cosmetic unsatisfaction. Cosmetic outcomes were analyzed and classified as excellent or good in 13 (39%) and 17 (52%) patients, respectively. No serious adverse events developed, except delayed epithelial healing in 3 cases.CONCLUSION:The cosmetic outcomes of the multiple noncontinuous transepithelial puncture technique for corneal tattooing were good. The safety of this method is higher than conventional procedures. This new procedure also provides improved cost-effectiveness and safety over current corneal tattooing techniques.     

角膜基质透镜在角膜溃疡或穿孔修复中的应用

目的：研究由全飞秒激光SMILE手术所得的角膜基质透镜作为角膜移植材料治疗角膜溃疡的临床疗效。

方法：回顾性病例研究。收集本院2017-01/06角膜溃疡患者6例6眼,其中细菌性、真菌性、深层异物伴感染各1例1眼,角膜穿孔3例3眼。采用由全飞秒激光SMILE手术所得的角膜基质透镜作为角膜移植的材料进行修复手术,确保植片与角膜层间无空气间隙。术后随访1～6(平均3.71±1.56)mo,观察手术前后视力、角膜移植物存活情况及术后并发症发生情况等。

结果：所有患者均在控制感染下顺利完成手术,无术中并发症。术后所有角膜植片透明。末次随访时,患者最佳矫正视力(best corrected visual acuity,BCVA)较术前明显改善(0.48±0.12 vs 1.50±0.08),差异具有统计学意义(P<0.01)。

结论：来源于全飞秒激光屈光手术的角膜基质透镜用于角膜溃疡修复是安全有效的,但植片的远期疗效尚需进一步观察。     

Anterior amorphous corneal opacity and corneal thinning

PurposeTo present the clinical features of four cases with bilateral anterior amorphous corneal opacity.MethodsA retrospective study in four patients with bilateral anterior amorphous corneal opacity was conducted. Examinations included visual acuity, keratometry, slit-lamp biomicroscopy, confocal microscopy, anterior segment optical coherence topography, and histology.ResultsThree female and one male patients (mean age, 52.3 ± 8.9 years) showed bilaterally oval, amorphous sheetlike corneal opacities with central depression and thinning. Superior limbal opacities were observed in two of these patients. The best-corrected visual acuity ranged from 20/50 to 20/400, and the mean of the keratometry was 39.81 ± 3.97 D (diopters). They had mild dry eyes. The anterior segment optical coherence topography demonstrated hyporeflective abnormalities in the anterior depressed stroma in these four patients. Confocal microscopy revealed large round cells at the epithelial layer in one patient, and amorphous opacities with some strand-shaped opacities in the anterior stroma in all four patients. The mean of the corneal endothelial cells density in the eight eyes was 1521 ± 402 cells/mm². Central corneal stromalysis occurred in three patients, and descemetocele developed in two eyes. One patient received penetrating keratoplasty and two underwent lamellar keratoplasty. The histology of the corneal specimen revealed edematous basal epithelial cells, focal collagen disorganization in the thin stroma, and wartlike excrescences in a thickened Descemet's membrane.ConclusionAnterior amorphous corneal opacity is a rare keratopathy and may be one kind of rare corneal degeneration or dystrophy. Corneal stromalysis may occur in hyporefrective amorphous opacities and progress to descemetocele.     

过夜配戴角膜矫形镜(OK镜)后角膜曲率及厚度变化

目的了解过夜配戴角膜矫形镜(OK镜)后早期角膜曲率、角膜厚度及屈光度的变化.方法14个中低度近视患者(平均屈光度-3.89±1.26D)25眼,随机分成2组分别配戴Dk值不同的角膜矫形镜BE角膜矫形镜组(7人12眼,Dk≈140);E&E角膜矫形镜组(7人13眼,Dk＝58).采用夜戴方式,平均配戴时间10.76±1.27小时.用Orbscan角膜地形图系统检查配戴1晚后角膜曲率、角膜厚度的改变.同时记录裸眼视力及屈光度的改变.结果配戴1晚后所有患者的中央及周边的角膜均增厚(P＜0.05).Orbscan角膜地形图SimK值在垂直轴及水平轴均变平(垂直轴△SimK0.94±0.56D,P＜0.001;水平轴△SimK0.996=0.68D,P＜0.001).裸眼视力平均提高0.4±0.24(P＜0.001).屈光度平均减少-1.85±0.82D(P＜0.001).Dk值不同的2组之间中央及颞上方位点角膜增厚程度的差别有统计学意义.结论过夜配戴角膜矫形镜后减低近视屈光度的效果明显.初次过夜配戴角膜矫形镜可导致角膜厚度轻度增加.     

美容性角膜覆盖术疗效观察

目的:评价美容性角膜覆盖术治疗角膜(角巩膜)葡萄肿的临床效果。方法:对22例角膜(角巩膜)葡萄肿患者进行了美容性角膜覆盖术治疗。术后对所有病例的临床效果进行了随访观察。结果:22例均治愈,残留部分视力的2例的视力术后有所改善(2例2眼从术前手动/眼前到术后指数/眼前)。22例无1例发生排斥,角膜覆盖片的颜色除第1例因缺乏经验术眼的颜色明显深于对侧正常或正常人群眼的颜色外,其它无1例褪色。结论:应用角膜覆盖术治疗角膜(角巩膜)葡萄肿22例(22眼)临床证明疗效确实、安全可靠。     

Determination of corneal volume from anterior topography and topographic pachymetry: application to healthy and keratoconic eyes

Purpose: To describe a method to measure corneal volume from topography and pachymetry, and test its clinical use on a sample of healthy human subjects and a case of circumscribed posterior keratoconus. Methods: Corneal curvature (PCT 200^® corneal topography system; Optopol Technology SA, Zawiercie, Poland) and ultrasonic topographic pachometry on 25 points (Ophthasonic^® A‐Scan/Pachometer III; Teknar Inc., St Louis, MO, USA) were measured on each of 12 young healthy corneas and one cornea suffering from circumscribed posterior keratoconus. Topography and pachymetry data were used to calculate the coordinates for the corresponding points on the posterior surface of the cornea. TableCurve 3D software (Systat Software Inc., Chicago, IL, USA) was used to fit a surface to those points measured. Integration of the surface fitted to the data points, corresponding to the anterior and posterior corneal surfaces, was used to calculate the volume underneath each of them. Subtraction of volumes underneath anterior and posterior surfaces, taking into account an axial offset equal to the central corneal thickness, rendered corneal volume for the central 6 mm of the cornea. Results: Central corneal thickness ranged from 520 to 630 μm for the healthy corneas. Corneal volumes for this sample analyzed averaged 18.66 ± 1.15 mm³ (range 17.25–20.53 mm³). For the posterior keratoconic cornea, the affected area was located at about 1.5–2 mm from the corneal center on the 135° hemimeridian of the right eye, observed through topographic pachymetry. Calculated corneal volume for the central 6 mm was 16.072 mm³, noticeably lower than those found in the sample without pathology, but within the range for corneas presenting with keratoconus. Conclusions: Corneal volume is a useful parameter for characterising dystrophic corneas and can aid in the detection of rare anomalies which are hardly detected with corneal topography and/or central corneal thickness evaluation. A potentially useful measure of corneal volume can be calculated from anterior corneal topography and topographic pachymetry data. Values obtained are in good agreement with previous studies using corneal tomography techniques. The methodology has been shown to have potential for retrospective analysis of data, or where no access is available to tomographical techniques.