组织工程角膜基质的体外构建及移植的实验研究

目的探讨利用组织工程技术体外构建角膜基质进行板层角膜移植的可行性和有效性。方法将猪角膜基质去细胞处理后制备成组织工程角膜基质载体;取幼兔角膜基质细胞体外培养,将其种植在载体上,体外构建成组织工程角膜基质,用PKH26荧光标记兔角膜基质细胞示踪角膜基质的构建;将16只兔的角膜基质内植入壳聚糖膜使之形成无菌性角膜溃疡,随机从16只兔中选8只,进行组织工程角膜基质移植;另外8只作为对照组,进行新鲜的同种异体兔板层角膜移植。术后对角膜进行裂隙灯、光学显微镜、透射电镜观察。结果体外构建的组织工程角膜的基质细胞具有活性,其结构与正常角膜基质相近。移植治疗无菌性角膜溃疡术后,1~2周有新生血管侵入组织工程角膜基质植片边缘,植片为灰白色半透明状;3~4周随着新生血管减退,组织工程角膜基质植片局部开始透明变薄;术后8~10周角膜溃疡完全修复,角膜恢复透明性,角膜神经可再生;观察最长达10月,角膜仍保持透明,无免疫排斥发生,与对照组疗效相同。结论体外构建的组织工程角膜基质无免疫原性、具有良好的生物相容性,可作为临床治疗角膜溃疡的移植材料。     

FK506滴眼液联合角膜移植术治疗复发性蚕蚀性角膜溃疡

目的：观察FK506滴眼液联合角膜移植术治疗复发性蚕蚀性角膜溃疡的疗效。方法：采用FK506滴眼液联合角膜移植术治疗复发性吞蚀性角膜溃疡患者9例（15只眼），对其中角膜溃疡＜2个象限角膜缘的2只眼，采用局部滴用0．1％，FD506滴眼液：角膜溃疡＞2个象限角膜缘的13只眼中，12只眼行球结膜切除及板层角膜移植术，1只眼因角膜溃疡穿孔，行穿透性角膜移植术，待术眼角膜上皮愈合后，局部滴用0．1％，FK506滴眼液，观察其疗效，同时对术中获取的角膜，结膜组织及房水，用酶联免疫分析法测定0．1％，FK506滴眼液在角膜，结膜组织及房水中的含量，对照组为板层角膜移植术联合0．1％地塞米松滴眼液治疗的12例复发性吞蚀性角膜溃疡患者。结果：滴用0．1％ FK506滴眼液后，角膜和结膜组织中FK506的含量为30－350ng/g，房水中未检测出FK506。9例（15只眼）吞蚀性角膜溃疡患者滴用0．1％，FD506滴眼液或联合角膜移植术治疗后，角膜溃疡均愈合，随访观察12－17个月，角膜溃疡无复发。视力提高＞2行者5只眼，对照组中，有7只眼的角膜溃疡复发，结论：局部应用0．1％ FK506滴眼液联合角膜移植术是治疗复发性吞蚀性角膜溃疡的有效方法。     

板层角膜移植术治疗真菌性角膜溃疡穿孔的临床观察

目的观察板层角膜移植术治疗真菌性角膜溃疡穿孔的临床疗效。方法对20例（20只眼）真菌性角膜溃疡穿孔患者,12只眼应用甘油冷冻保存的角膜进行板层角膜移植术,8只眼进行结膜瓣遮盖术。结果板层角膜移植术组疗效明显优于结膜瓣遮盖组,12只眼角膜穿孔均得到控制。结论在没有新鲜角膜材料的情况下,应用冷冻的角膜对真菌性角膜溃疡穿孔的患者做板层角膜移植术有一定的临床价值。     

兔角膜重度碱烧伤后异种猫前板层角膜移植重建角膜眼表

目的研究猫新鲜前板层角膜移植治疗兔右眼角膜重度碱烧伤的疗效。方法将16只5个月龄的健康雄性新西兰白兔右眼制作重度角膜碱烧伤模型,随机分成A、B两组,每组各8只兔,其中A组为猫(4只)→兔异种前板层角膜移植组,植片直径为11.0mm,植床直径为10.0mm;B组未行手术。A组兔术后及B组造模后用500g.L-1葡萄糖及复方氯霉素滴术眼直至处死,记录两组疗效指数,并分别于术后1个月、2个月、3个月取材进行术眼角膜病理学及扫描电镜检查。结果3个月内A组8只角膜碱烧伤兔经异种猫前板层角膜移植后,7只移植角膜保持透明,1只出现局限性角膜混浊和角膜新生血管;而B组未手术的8只兔中出现角膜白斑、新生血管4只,角膜溃疡、穿孔3只,角膜溶解穿孔1只,并且有4只发生睑球粘连等并发症。A、B两组治疗后1个月、2个月、3个月平均疗效指数差异均有统计学意义(均为P<0.05)。A组透明角膜中组织学及扫描电镜下的结构特征清晰;B组兔角膜中有明显的角膜水肿和炎性细胞浸润,组织学及扫描电镜下的结构特征显示细胞连接混乱、消失。B组扫描电镜还提示随着时间的延长,上皮细胞部分生长、增多,细胞连接差,角膜上皮排列紊乱;角膜内皮细胞数量明显减少或无,...     

板层角膜移植联合羊膜层间植入治疗重症蚕蚀性角膜溃疡

目的探讨板层角膜移植联合保存人羊膜移植重建角膜基质和眼表结构,治疗重症蚕蚀性角膜溃疡的疗效和安全性。方法重症蚕蚀性角膜溃疡患者5例(5眼),具疼痛性、进行性角膜溶解的临床特征,常规治疗方法疗效差,病变区域累及角膜缘6～12个钟点(平均9个钟点)范围。手术前全身和局部应用皮质类固醇。切除角膜缘处5mm宽的结膜和筋膜组织暴露巩膜。仔细的清除溃疡的基底部和边缘部组织,在坏死组织的表面和新生血管区域行烧灼,单层羊膜覆盖在溃疡区域的表面,10-0尼龙线间断缝合固定。在角膜穿孔的部位,移植多层羊膜以修复角膜基质层。在深层溃疡区域,行板层角膜移植。术后,局部应用人工泪液、抗生素眼液和类固醇眼液。术后观察前房深度、再上皮化速度、炎症状况、角膜基质厚度稳定状况、溃疡复发情况和视力。术后7d每日行常规检查,以后每周复查1次。随访期4～15(平均8.6)月。结果 手术获得了良好的疗效。术后自觉症状消失,眼表炎症明显减退,角膜溃疡的表面上皮稳定愈合,新生血管减少,获得了平滑、湿润的眼表。板层角膜植片平复半透明。未见溃疡复发。术后视力无明显下降。无手术并发症发生。结论 角膜板层移植联合羊膜移植可用于治疗重症蚕蚀性角膜溃疡。     

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。     

不规则移植片板层角膜移植治疗蚕蚀性角膜溃疡

目的：探讨不规则移植片板层角膜移植联合术后1％环孢霉素A滴眼治疗蚕蚀性角膜溃疡的效果及对视力的影响。方法：应用不规则移植片板层角膜移植治疗蚕蚀性角膜溃疡9例10只眼,术中根据溃疡侵犯角膜的形态切除病变角膜组织及邻近的球结膜和筋膜组织,按照植床的形态制作之相吻合的板层移植片行板层角膜移植术术,术后应用1％环孢霉素A滴眼6－8个月,结果术后随访1－5年,未见复发病例,术后6个月,与术前视力比较,大部分病例视力不变或提高。结论：不规则移植片板层角膜移植术联合术后应用1％环孢霉素A滴眼治疗蚕蚀性角膜溃疡效果肯定,只要植片与植床吻合,疑线松紧适宜,不会导致明显和光而影响视力。     

板层角膜移植术治疗真菌性角膜溃疡效果的探讨

目的探讨板层角膜移植治疗真菌性角膜溃疡的临床效果。方法12例（12眼）真菌性角膜溃疡角膜感染灶面积均大于8mm直径。术中彻底切除病灶组织，用冷冻保存角膜材料行板层移植。结果12例（12眼）术后均有效控制了感染，观察半年，未见复发。视力有不同程度的提高。结论板层角膜移植可有效治疗真菌性角膜溃疡。     

治疗性角膜移植的临床应用

治疗性角膜移植的许多新术式,如角膜层间填垫术、深板层角膜移植术、后板层内皮角膜移植、角膜上皮干细胞移植、全角膜上皮联合后板层内皮角膜移植、蕈状瓣角膜移植术业经临床验证,能使濒临穿孔或业已穿孔的角膜溃疡、复发性单纯疱疹病毒角膜基质炎、圆锥角膜、内皮衰竭引发的大泡性角膜病变、重症眼表烧伤等致盲性角膜病获得康复.通过回顾性研究,对上述相关术式的适应证、手术要点、疗效作了详尽的讨论.     

部分板层角膜移植联合层问羊膜移植治疗角膜穿孔

目的 探讨层间羊膜移植联合部分板层角膜移植治疗角膜溃疡穿孔的临床疗效。方法对4例角膜溃疡穿孔患者,行角膜溃疡板层切除、层间羊膜移植联合部分板层角膜移植术。术后利用共焦显微镜和裂隙灯显微镜进行观察,随访1～7个月。结果术后3例裸眼视力得到提高,1例裸眼视力下降,但所有病例的矫正视力都有提高。未见双前房、免疫排斥反应等并发症。结论该手术是一种有效的治疗周边角膜穿孔的方法。     

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。     

Influence of corneal astigmatism, corneal curvature and meridional differences on corneal hysteresis and corneal resistance factor

Background: This study investigated the influence of corneal astigmatism, corneal curvature and meridional differences on corneal hysteresis (CH) and the corneal resistance factor (CRF) in a group of normal Chinese persons. Methods: Ninety‐five participants were recruited and data from the eye with higher corneal astigmatism were analysed. The anterior corneal curvature was measured by corneal topography. The Goldmann‐correlated intraocular pressure (IOPg), corneal‐compensated intraocular pressure (IOPcc), CH and CRF at different meridians (default horizontal position, 10°, 20° and 30° along the superotemporal and inferonasal meridians) were obtained from an ocular response analyser. The corneal powers at these specific meridians also were calculated. Results: At the default position, the IOPg and CRF had weak correlations with corneal astigmatism, while the IOPcc and CH were not significantly correlated with corneal astigmatism. Both the IOPg and IOPcc were measured significantly higher at the default position. The CH and CRF were lower at the default position but the difference in the CRF from obliquity could not reach statistical significance. The CH was not significantly correlated with the corneal power at all meridians. The CRF correlated with the corneal power only at 30° superotemporal. Conclusion: Corneal astigmatism and head tilt did not have much effect on the measurement of CH and the CRF, both of which were lowest along the horizontal meridian. Clinically, the difference was small. The influence of corneal power on CH and the CRF was minimal.     

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.     

角膜生物力学与光密度相关性研究

目的:探究角膜生物力学与角膜光密度的相关性。方法:前瞻性研究。选取2019-03/06在云南省第二人民医院拟行角膜屈光手术术前检查的患者为研究对象。采用Pentacam HR眼前节分析系统进行角膜光密度测量,以角膜顶点为中心,分为0~2mm、> 2~6mm、> 6~10mm直径范围3个区域,以角膜厚度分为前、中、后3层。选取Pentacam HR中角膜最薄点厚度值纳入研究。采用Corvis ST角膜生物力学分析仪测量,相关参数包括第一次压平的长度(AP1L)和速率(AP1V)、第2次压平的长度(AP2L)和速率(AP2V)、最大凹陷时顶点距离(PD)、曲率半径(HCR)和形变幅度(DA)。运用Pentacam&Corvis ST生物力学联合诊断平台软件综合分析检查结果,得出综合角膜生物力学参数(CBI)以及其它独立参数包括硬度参数(SP)、综合半径(IR)、Ambrosio相关厚度-水平方向(ARTh)、形变幅度比(DAR)。各区域光密度间差异采用方差分析,角膜生物力学各项参数与各区域光密度的相关性采用Pearson或Spearman分析。结果:不同直径范围、不同层面间光密度有差异(F=35.101,P<0.01;F=1002.897,P<0.01),CBI与独立生物力学参数中AP2L、AP2V、PD、DA、SP、IR、ARTh、DAR具有相关性(rs=-0.502,P<0.01;rs=-0.457,P=0.001;rs=0.428,P=0.002;rs=0.539,P<0.01;rs=-0.687,P<0.01;rs=0.716,P<0.01;rs=-0.728,P<0.01;rs=0.750,P<0.01)。CBI与角膜0~2mm范围内光密度呈正相关(r=0.343,P=0.015)。0~2mm范围内光密度与独立生物力学参数中AP2L、IR、ARTh、DAR有相关性(rs=-0.298,P=0.035;rs=0.368,P=0.009;rs=-0.419,P=0.002;rs=0.493,P<0.01)。结论:角膜中央区域光密度与角膜生物力学具有显著的关联,临床中可以通过光密度和生物力学对角膜健康状况进行综合评价。     

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。     

角膜内皮炎的角膜内皮细胞形态改变

目的探讨角膜内皮炎对患者中央角膜内皮细胞形态的影响。方法用EM-1000型接触式角膜内皮镜对10例单眼角膜内皮炎愈后4-12周的患者的双眼分别摄取中央角膜内皮像并对其图像进行电脑分析。患眼作为实验组。健眼作为对照组。观察其角膜内皮细胞密度。六角形细胞的百分比及异形性的变化。用计量资料配对设计的2样本均数的t检验进行统计分析（双侧检验，P〈0．01为统计学有差异）。结果在10例临床诊断角膜内皮炎愈后的患者中，中央角膜内皮细胞密度实验组平均为（1981±181）／mm^2。对照组平均为（2284±315）／mm^2。六角形细胞比例实验组平均为34％，对照组平均为43％．变异系数实验组平均为53％，对照组平均为45％．以上各项观察指标实验组与对照组之间在统计学上都存在显著性差异（P〈0．01）。结论角膜内皮炎对角膜内皮细胞造成损伤，导致严重的形态改变，在临床工作中应给予充分重视。[眼科新进展2007；27（2）：138-139]     

Alcohol delamination of the corneal epithelium for recurrent corneal erosion syndrome

AIM: To evaluate the outcomes of alcohol delamination (ALD) of the corneal epithelium for the treatment of recurrent corneal erosion syndrome (RCES) and to implement a standardized treatment protocol for this condition utilizing evidence based practice and the findings of an internal audit. METHODS: A retrospective analysis of 42 eyes of 40 patients diagnosed with RCES who were treated with ALD between January 2006 and March 2016 was conducted. Patients had 20% alcohol applied to the cornea with the use of a well for 40s. Patients were reviewed one week later in the Outpatient Department. Outcome criteria were established based on standards from other studies in the medical literature. These included, a treatment success rate of at least 72% (defined as complete resolution of symptoms one month after treatment), a postoperative complication a rate of <5% (mainly infective keratitis, and subepithelial haze), and the absence of any detrimental effect on visual acuity in ≥95% of patients. RESULTS: The mean age at the time of ALD was 41.17±13.44y. Patients were followed for an average of 12.8±15.65mo. The majority were female (52.5%, n=21) and the majority of eyes treated with ALD were left eyes (62.9%, n=26). Trauma was the primary aetiology in our study population. Treatment was successful in 73.8% (n=31) of eyes and in 75% (n=30) of patients. Recurrence occurred in 26.2% of eyes at a mean of 10.41±12.63mo post treatment. CONCLUSION: ALD is an efficacious and cost-effective primary surgical intervention for RCES.     

Orbscan-II角膜地形图仪与超声角膜测厚仪角膜测厚结果的对比分析

目的:探讨Orbscan-II角膜地形图仪与角膜测厚仪对角膜测厚的结果差异的统计学及临床意义。方法:选115例204眼(右104眼,左100眼)分别行Orb-scan-II及超声测厚法测量角膜厚度,经统计学检验后进行分析。结果:204眼Orbscan-II所得结果为527.20±37.83,超声测厚法所得结果为525.25±34.83,行配对t检验,t=2.407,P=0.017,认为两种测量方法结果的差异有统计学意义。进一步将角膜厚度分4组进行配对t检验,角膜厚度<500μm时,Orbscan-II测厚(478.36±14.47)与超声测厚(478.3±20.15)差异无统计学意义,角膜厚度≥500,<550μm时,Orbscan-II测厚(527.88±17.32)与超声测厚(525.32±12.92)差异有统计学意义(P=0.027),角膜厚度≥550,<600μm时,Orbscan-II测厚(571.24±14.89)与超声测厚(568.04±13.82)差异有统计学意义(P=0.022),角膜厚度≥600μm时,Orbscan-II测厚(615.33±8.50)与超声测厚(615.33±14.29)差异无统计学意义。结论:传统的超声测厚与Orbscan-II对角膜测厚结果有一定差异,并具统计学意义,但在临床意义上二者仍有较好的相符性。     

配戴软性角膜接触镜对角膜中央厚度影响的临床研究

目的:探讨配戴软性角膜接触镜对中央角膜厚度的影响。方法:应用超声角膜测厚仪检测中央角膜厚度,在我院近视患者中随机抽取配戴软性角膜接触镜者及不戴镜者各100例200眼,做统计分析。再在近视患者中按戴镜时间<1a,1～3a,3～5a,5～7a,≥7a分组,每组随机抽取60例120眼,做统计分析。结果:配戴软性角膜接触镜者与不戴镜者的角膜厚度比较,差异有统计学意义(P<0.05)。戴镜时间<1a,1～3a,3～5a,5～7a者的角膜厚度比较,差异有统计学意义(P<0.05)。结论:配戴软性角膜接触镜者平均中央角膜厚度薄于不戴角膜接触镜者,并且配戴软性角膜接触镜的时间越长中央角膜厚度越薄。     

Construction of a full-thickness human corneal substitute from anterior acellular porcine corneal matrix and human corneal cells

AIM: To construct functional human full-thickness corneal replacements. METHODS: Acellular porcine corneal matrix (APCM) was developed from porcine cornea by decellulariztion. The biomechanical properties of anterior-APCM (AAPCM) and posterior-APCM (PAPCM) were checked using uniaxial tensile testing. Human corneal cells were obtained by cell culture. Suspending ring was designed by deformation of an acupuncture needle. MTT cytotoxicity assay was used to check the cytotoxicity of suspending ring soaking solutions. A new three-dimensional organ culture system was established by combination of suspending ring, 48-well plate and medium together. A human full-thickness corneal substitute was constructed from human corneal cells with AAPCM in an organ coculture system. Biochemical marker expression of the construct was measured by immunofluorescent staining and morphological structures were observed using scanning electron microscopy. Pump function and biophysical properties were examined by penetrating keratoplasty and follow-up clinical observations. RESULTS: There were no cells in the AAPCM or PAPCM, whereas collagen fibers, Bowman’s membrane, and Descemet’s membrane were retained. The biomechanical property of AAPCM was better than PAPCM. Human corneal cells grew better on the AAPCM than on the PAPCM. There was no cytotoxicity for the suspending ring soaking solutions. For the constructed full-depth human corneal replacements keratocytes scattered uniformly throughout the AAPCM and expressed vimentin. The epithelial layer was located on the surface of Bowman’s membrane and composed of three or four layers of epithelial cells expressing cytokeratin 3. One layer of endothelial cells covered the stromal surface of AAPCM, expressed Na+/K+ATPase and formed the endothelial layer. The construct was similar to normal human corneas, with many microvilli on the epithelial cell surface, stromal cells with a long shuttle shape, and zonula occludens on the interface of endothelial cells. The construct withstood surgical procedures during penetrating keratoplasty. The corneal transparency increased gradually and was almost completely restored 7d after surgery. CONCLUSION: AAPCM is an ideal scaffold for constructing full-thickness corneal replacement, and functional human full-thickness corneal replacements are successfully constructed using AAPCM and human corneal cells.