A review of the evidence for in vivo corneal endothelial regeneration

Human corneal endothelium has long been thought to be a nonmitotic cell layer with no endogenous reparative potential. Pathologies that damage endothelial function result in corneal decompensation and, if untreated, blindness. The mainstay of treatment involves partial or complete corneal replacement, amounting to 40% of all corneal transplants performed worldwide. We summarize the case reports describing complications postoperatively in the form of (sub)total graft detachment and those resulting in postoperative bare stroma. Complications during cataract and glaucoma surgeries leading to an uncovered posterior cornea are also included. We discuss the newer treatment strategies that are alternatives for current Descemet membrane endothelial keratoplasty and Descemet stripping automated endothelial keratoplasty, including partial grafts and stripping of the diseased cell layer. In more than half of the cases reviewed, corneal transparency returned despite incomplete or no corneal endothelial cell transplantation. We question the existing paradigm concerning corneal endothelial wound healing in vivo. The data support further clinical study to determine the safety of simple descemethorexis in central endothelial pathologies, such as Fuchs endothelial corneal dystrophy, where presence of healthy peripheral cells may allow successful corneal recompensation without the need for donor cells.     

微环境对角膜内皮作用机制的研究进展

角膜内皮作为角膜中代谢最为活跃的一层,在维持角膜透明性中发挥重要作用.角膜内皮微环境则是揭示角膜内皮疾病机制和体外培养角膜内皮进而解决角膜内皮移植供体匮乏的关键,其包括了普遍存在的,对细胞有直接作用的生长因子、营养因子、炎性因子及干性因子等各种细胞因子.同时因角膜内皮位置特异性所形成的高氧化环境、眼压等物理应力及相邻组织,如晶状体、角膜后弹力层、房水等与角膜内皮的相互作用也是微环境中的重要因素     

角膜内皮细胞治疗研究进展

人角膜内皮细胞(HCECs)是一种有丝分裂后的单层内皮细胞,因此,其在体内和体外的增殖能力十分有限。HCECs在严重受损的情况下会发生内皮失代偿,极易引起失明。目前,唯一有效的治疗方法是使用含健康角膜内皮的供体植片进行角膜移植。因此,世界范围内供体材料的严重短缺推动了对角膜内皮替代来源的研究。随着HCECs的细胞培养研究的不断开展,细胞治疗为角膜内皮失代偿提供了希望。本文对角膜内皮细胞治疗方面的最新研究进展进行综述。     

羟乙基淀粉130/0.4对器官培养法保存兔角膜的脱水作用

目的：研究羟乙基淀粉的最新剂型HES 130/0.4对器官培养保存角膜的持续脱水效果。

方法：20对配对兔角膜,一半于含10% HES 130/0.4的ACF培养液中保存28d作为实验组,不再单独脱水; 另一半于ACF培养液中保存28d后再葡聚糖T500脱水48h作为对照组。内皮细胞活性和植片质量评估指标包括：内皮细胞计数、角膜厚度和含水量、角膜透明度和后弹力层皱褶程度、内皮层中肌动蛋白微丝(filament actin,F-actin)的表达,及透射电镜下内皮细胞超微结构的变化。

结果：保存结束时,实验组植片明显较薄,角膜透明度和皱褶程度也优于对照组,内皮细胞密度为2371±159个/mm²。对照组继续脱水后,角膜厚度、含水量和透明度与实验组间差别变小,内皮细胞密度却降至2138±182个/mm²。免疫印迹法证实F-actin在两组内皮层都有表达,实验组的F-actin表达水平更高。电镜下实验组的细胞超微结构改变较小。

结论：HES 130/0.4的细胞毒性小,可成为器官保存液中的持续添加组分,不仅避免角膜过度水肿,还简化了保存程序,减轻了感染风险,有希望成为新型角膜脱水剂。     

水通道蛋白1在人角膜内皮细胞的表达

目的 研究水通道蛋白1(water channel protein1,AQP1)抗体在人角膜中的表达及水转运机制。方法 运用免疫组织化学法测定人角膜中AQP1的表达。结果 在角膜内皮细胞和基质细胞中有AQP1的表达，而角膜上皮细胞不表达AQP1。结论 本实验证实了AQP1在角膜内皮的表达，为角膜的水转运及角膜水肿机制的研究提供了分子生物学基础。     

Glaucoma-associated corneal endothelial cell damage: A review

The corneal endothelium is critical in maintaining a healthy and clear cornea. Corneal endothelial cells have a significant reserve function, but preservation of these cells is paramount as they have limited regenerative capacity. Glaucoma is a prevalent disease, and damage to the corneal endothelium may be caused by the disease process itself as well as by its treatment. The mechanisms involved in glaucoma-associated damage to the corneal endothelium need further investigation. Understanding how glaucoma and glaucoma surgery impact the endothelium is important for protecting corneal clarity and visual acuity in all glaucoma patients, including those undergoing corneal transplant. We will discuss a range of identified factors that may impact corneal endothelial cell health in glaucoma, including intraocular pressure, glaucoma medications, surgical glaucoma management, mechanical forces, and alterations in the aqueous environment.     

不同方法获取Descemet膜-角膜内皮细胞复合体的效果比较

目的 评价不同方法获取Descemet膜-角膜内皮细胞复合体(DM CECs)的效果.方法 将20只兔(40只眼)根据手术方法的不同随机分成4组:镊子剥离组、水分离组、大气泡法组和机械分离组,每组10只眼.通过活体染色和组织切片分析评价DM CECs.结果 成功获取DM CECs镊子剥离组2眼,水分离组8眼,大气泡法组5眼,机械分离法组3眼,4个组之间的成功率差异有统计学意义(P<0.05).水分离法组(98.03 ±2.18)%和大气泡法组(97.14 ±3.04)%内皮细胞存活率(ESR)明显高于镊子剥离法组(80.16 ±5.21)%和机械分离法组(71.56 ±9.57)%(F=436.04,P<0.05).苏木精-伊红染色显示水分离法和大气泡法对内皮细胞损伤小.结论 水分离法或大气泡法是安全、有效地获取DM CECs的技术.     

白内障术后角膜病变治疗的新进展

白内障患者晶状体超声乳化术后的角膜水肿、干眼、角膜内皮功能失代偿致大泡性角膜病变等严重影响着患者满意度和视觉质量。长期以来,穿透性角膜移植是角膜内皮功能失代偿患者视力恢复的有效方法,但角膜供体缺乏及移植后角膜内皮细胞密度降低等问题仍然无法解决。近年来,角膜内皮移植、局部角膜后弹力层撕除、人工培养角膜内皮细胞植片的移植、Rho激酶抑制剂调节角膜内皮细胞增殖等方面的进展和新手段,为白内障术后角膜内皮功能失代偿患者的治疗带来新思路。     

A COMPARISON OF CORNEAL ENDOTHELIAL MORPHOLOGY IN CORNEA GUTTATA, FUCHS' DYSTROPHY AND BULLOUS KERATOPATHY

Changes on corneal endothelial specular microscopy were compared in 14 patients with cornea guttata, 4 patients with Fuchs' corneal dystrophy and 19 patients with various forms of bullous keratopathy. The patients with cornea guttata showed well marked guttae 1 to 6 endothelial cells in diameter in the endothelial mosaic and in the relief mode while the endothelial mosaic was usually otherwise within normal limits. In 2 patients with Fuchs' dystrophy the endothelium could be examined, showing gross guttae but a few areas of relatively normal endothelial cells. The unaffected eye of 3 other patients snowed findings similar to cornea guttata, but with some reduction in endothelial cell count in 2 patients. The patients with bullous keratopathy fell into 2 groups, one with gross reduction in cell count in a markedly abnormal endothelial cell mosaic, the other a mixed group with moderate reduction in cell count and numerous guttae. Some miscellaneous cases included one of aphakic peripheral bullous keratopathy, one associated with cyclitis and aphakia and 2 with idiopathic non-surgical bullous keratopathy. We believe the corneal endothelium is not grossly abnormal away from the guttae in Fuchs' dystrophy, but the gross guttata formation determines the endothelial dysfunction.     

无缝线深板层角膜内皮移植术的研究进展

角膜内皮失代偿可严重影响视力,以往多采用的穿透性角膜移植手术(PKP)具有术后高度散光、移植片排斥等并发症,严重限制了术后视力的提高.如果能够单纯地进行后弹力层和内皮细胞层的移植,将减少手术操作所带来的受体角膜损伤并能更好地恢复术后视力.近年来,无缝线深板层角膜内皮移植手术的优点已逐渐获得公认;其中深板层角膜内皮细胞移植手术(DLEK)手术难度较大,限制了其在临床的广泛开展.而通过剥除受体角膜的后弹力层和内皮层来制作植床的角膜内皮移植手术即角膜后弹力层剥除内皮细胞移植手术(DSEK)已经取得了较好的手术效果.本文归纳、分析了DSEK的手术方法、效果及并发症等.     

bFGF和PDGF对家兔角膜内皮损伤修复作用的研究

目的观察碱性成纤维细胞生长因子(bFGF)与血小板源性生长因子(PDGF)联合应用时对家兔角膜内皮冷冻损伤后修复的作用。方法48只家兔角膜冷冻损伤后随机分为4组,分别为对照组、bFGF组、PDGF组及联合应用bFGF与PDGF组,于不同时间点分别观察角膜内皮细胞数、六边形细胞百分数、角膜厚度、角膜内皮缺损面积。结果bFGF与PDGF联合应用组应用后的角膜内皮细胞数最多、六边形细胞比例最大、角膜厚度最接近正常、角膜内皮缺损面积最小;联合应用组的角膜内皮恢复的速度和质量最高,与其他3组相比差异有统计学意义。结论bFGF与PDGF联合应用时能促进角膜内皮细胞的增生和移行,对加速角膜内皮损伤后的修复具有协同作用。     

Research progress on the negative factors of corneal endothelial cells proliferation

The human corneal endothelium forms a boundary layer between anterior chamber and corneal stoma. The corneal endothelial cells are responsible for maintaining cornea transparency, which is very vital for our visual acuity, via its pump and barrier functions. The adult corneal endothelial cells in vivo lack proliferation in response to the cell loss caused by outer damages and diseases. As a result, in order to compensate for cell loss, corneal endothelial cells migrate and enlarge while not via dividing to increase the endothelial cell density. Therefore, it is not capable for corneal endothelium to restore the corneal clarity. Some researches have proved that in vitro the corneal endothelial maintained proliferation ability. This review describes the current research progress regarding the negative factors that inhibit proliferation of the corneal endothelial cells. This review will mainly present several genes and proteins that inhibit the proliferation of the corneal endothelial cells, of course including some other factors like enzymes and position.     

大泡性角膜病变的临床治疗及研究进展

各种原因引起的角膜内皮细胞损害,致角膜内皮失代偿,产生角膜基质水肿、上皮下水肿,最终形成的大泡性角膜病变（bullous keratopathy,BK）已成为常见的致盲性角膜病。而BK并非一种独立的疾病,往往与其他多种眼病同时存在,使其更加不易治疗。近年来,除了临床常用的药物及传统的手术方式外,学者们还积极开发新的角膜层间术式,拓展羊膜在手术中的应用及其复合术式;对角膜内皮细胞移植术及体外培养角膜内皮细胞移植的不断探索和发展,在临床和研究上都取得了一定的进展。本文就BK的临床治疗和研究进展作一综述。     

两种常见的角膜深板层内皮移植术的临床对照研究

目的 对照研究两种常见的角膜深板层内皮移植术的适应证、技术特点、手术效果、并发症及其处理.方法 对A组6例角膜内皮失代偿患者,实施了在角膜瓣下进行角膜深板层内皮移植术.B组9例同样患者实施了在角膜基质袋内进行的角膜深板层内皮移植术.术后追踪观察并发症以及最佳矫正视力、角膜散光、角膜厚度和角膜内皮细胞密度.结果 A组,术后6个月,5例患者视力明显提高.平均散光(2.16±0.89)D.平均角膜厚度(546±58.8)μm.平均内皮细胞密度为(2480±214)个/mm2.B组,随访6个月,所有患者最佳矫正视力不同程度地提高.平均散光(1.19±0.48)D.平均角膜厚度(533±51.7)μm.平均内皮细胞密度为(2124±278)个/mm2.结论 两种角膜深板层内皮移植术均无严重的难以克服的并发症.在角膜瓣下进行的角膜深板层内皮移植术,比较适用于有晶体眼.在角膜基质袋内进行的角膜深板层内皮移植术对于早期的角膜内皮失代偿患者,术后屈光效果较好.适用于无晶状体眼、人工晶状体眼和同时需要进行白内障手术的患者.两种术式均适合我国患者的特点.     

角膜内皮移植术

角膜内皮移植是一种新的治疗角膜内皮病变的手术方法,主要是采用健康的角膜内皮片替代病变内皮层.该方法因为几乎不改变患者的角膜曲率和屈光状态,损伤小,视力恢复快,近年来在临床上逐渐被推广应用,取得较好愈后效果.该方法是角膜移植手术不断精细化和向屈光性手术转化的具体体现.角膜内皮移植术后良好的屈光效果和可能的低排斥率,使其有望成为角膜内皮病变治疗的重要方式.但是,目前内皮移植手术仍然需要完善,改进植入方法以减少脱片率和内皮细胞损失率,利用飞秒激光等进一步提高屈光效果,以及观察术后免疫排斥率是今后眼科学者的研究重点.     

Surgical strategies to improve visual outcomes in corneal transplantation

The recent years have brought about a sea change in the field of corneal transplantation with penetrating keratoplasty being phased to newer lamellar keratoplasty techniques for a variety of corneal pathology. Improved and innovative surgical techniques have allowed selective replacement of diseased host corneal layers with pre-prepared healthy donor corneal lamellae for anterior corneal disorders such as keratoconus and posterior corneal disorders such as Fuch''s corneal endothelial dystrophy. The results of lamellar techniques are encouraging, with rapid visual rehabilitation and vastly reduced risk of immune-mediated transplant rejection. The techniques of deep anterior lamellar keratoplasty and Descemet''s stripping endothelial keratoplasty (DSAEK) continue to evolve with advent of femtosecond lasers and newer concepts such as pre-conditioned donor corneas for Microthin DSAEK and Descemet''s membrane keratoplasty. This review describes the current developments in lamellar keratoplasty, including the futuristic approach using cell therapy to restore vision in corneal blindness.     

夜戴型角膜塑形镜对青少年近视患者眼球参数的短期影响

目的：：评价青少年近视患者配戴夜戴型角膜塑形镜后6 mo角膜内皮细胞密度、中央角膜厚度、角膜曲率和眼轴长度的变化。方法：回顾性病例研究。利用超声诊断仪、非接触角膜内皮显微镜和角膜地形图仪,测量和观察44例86眼7~17岁青少年近视患者配戴夜戴型角膜塑形镜前和配戴后6 mo的角膜中央厚度、角膜内皮细胞密度、中央角膜厚度、角膜曲率和眼轴长度的变化和相关性。结果：配戴夜戴型角膜塑型镜6 mo后,白天裸眼视力稳定在1.0~1.2,无明显眼部并发症。角膜内皮细胞密度稍有减少,但与戴镜前相比差异无统计学意义(P>0.05)。角膜曲率及前房深度无明显变化。角膜中央厚度变薄及眼轴长度仍有增长,差异有统计学意义(P<0.05)。角膜内皮细胞密度与眼轴长度具有相关性(右眼：P=0.005,左眼：P=0.013),中央角膜厚度与眼轴长度具有相关性(右眼：P=0.019,左眼：P=0.006)。结论：配戴夜戴型角膜塑型镜6mo,眼轴长度依然有增长,角膜内皮细胞密度降低,中央角膜厚度变薄。     

Gene transfer to ovine corneal endothelium

Purpose : Modification of a donor cornea by gene therapy has potential to modulate irreversible rejection, the major cause of corneal graft failure. The sheep is a useful model for the human in this respect, as ovine endothelial cells are amitotic. The aim of the study was to investigate the ability of various non‐viral and viral agents to transfer a reporter gene to ovine corneal endothelium. Methods : The non‐viral agents Transfectin‐10, Transfectin‐20, Transfectin‐50, SuperFect, Effectene and CLONfectin were used to deliver the reporter gene, Escherichia coli lacZ, to ovine corneal endothelium in vitro. A Herpes simplex virus‐1 and an adenoviral vector each encoding E. coli lacZ were similarly tested. Infected corneas were organ‐cultured for up to 7 days in vitro to allow transfection efficiency, duration of gene expression and toxicity attributable to each vector to be compared. Results : Scattered single or clusters of endothelial cells expressing the reporter gene were observed after transfection with CLONfectin, Transfectin‐10, Transfectin‐20 and Transfectin‐50. SuperFect and Effectene were virtually in‐effective. At best, the absolute number of infected cells per endothelial monolayer after 3 or 7 days of organ culture was estimated as < 0.01%. The Herpes simplex virus‐1 vector also failed to transduce ovine corneal endothelium efficiently. In contrast, transfection rates of up to 70% of endothelial cells were observed with the adenoviral vector. Conclusion : Non‐viral vectors and Herpes simplex virus‐1 are unlikely to be suitable for gene therapy of corneal endothelium, because the efficiency of transfection is low compared with the rates achieved with adenoviral vectors.     

Corneal Endothelial Transplantation

Patients with visually significant corneal edema, secondary to endothelial dysfunction, before the development of scarring or vascularization, need only have the corneal endothelium replaced to restore corneal clarity. This fact, plus the lack of consistently available donor material, prompted us to evaluate tissue cultured corneal endothelium (TCCE) as a donor source. We have shown that TCCE, when transplanted, can regain normal morphology and physiologic function. To accomplish practical use of autologous stroma, a transparent gelatin membrane which can serve as substrate for endothelial growth in tissue culture, has been developed. This cellular membrane has been transplanted successfully in rabbits with good functional results. It is hoped that ultimately this technique can be developed for routine use in man.     

组织工程角膜三维构建的实验研究

目的以人胚胎干细胞（hESC）诱导细胞为种子细胞,以脱细胞猪角膜基质（APCM）为支架三维构建生物工程角膜,以期用于穿透性角膜移植,解决角膜供体极度匮乏的难题。方法实验研究。无菌条件下将新鲜猪角膜组织置于0.5% SDS溶液中4 ℃脱细胞 24 h,获取APCM。将hESCs与人角膜基质细胞通过Transwell共培养5 d,获取眼周间充质干细胞（POMPs）,再于人晶状体上皮细胞源性条件培养基继续培养14 d获取角膜内皮样细胞并进行鉴定和筛选纯化。将纯化后扩增的角膜内皮样细胞接种于APCM构建角膜内皮植片,并移植入角膜内皮功能失代偿动物模型进行泵功能评估;采用人角膜缘干细胞（LSCs）来源的条件培养基培养hESCs 12 d,诱导其分化人角膜上皮样细胞并筛选鉴定,将其与APCM构建的角膜上皮植片移植于LSC失代偿动物模型的角膜缘,观察其眼表修复能力。结果诱导的人角膜内皮样细胞表达内皮细胞相关标记物vimentin、N-cadherin、Na+/K+ATP酶和ZO-1。构建的角膜内皮植片能够促使角膜内皮功能失代偿动物的角膜逐渐恢复透明。构建的角膜上皮细胞植片具有4～5层细胞复层结构,类似于正常角膜上皮,且能够一定程度上修复LSC失代偿动物模型眼表。结论采用hESCs诱导分化来源的细胞与APCM构建的人角膜内皮植片和人角膜上皮植片具有类似于正常角膜的功能,为全层生物角膜的构建提供了良好的实验和理论基础,具有良好的临床应用前景。