角膜内皮细胞移植

角膜内皮细胞移植是治疗角膜内皮疾病很有潜力的一种方法,目前仍停留于动物实验阶段,主要难点是供体角膜内皮细胞的培养和维持、细胞载体和手术技巧等问题,本文围绕这些问题进行综述.     

角膜内皮细胞移植

角膜内皮细胞移植是治疗角膜内皮疾病很有潜力的一种方法,目前仍停留于动物实验阶段,主要难点是供体角膜内皮细胞的培养和维持、细胞载体和手术技巧等问题,本文围绕这些问题进行综述。     

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)     

体外培养角膜内皮移植的研究进展

随着角膜内皮细胞体外培养扩增技术不断完善，以及近年后（深）板层角膜内皮移植技术的兴起，以各种载体进行培养角膜内皮细胞进行后板层移植，以代替穿透性角膜移植的研究具有广阔的应用前景。本文就角膜内皮细胞的培养、移植载体的选择和移植技术的发展加以综述，并对目前仍需解决的问题及其前景进行展望。     

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。     

体外培养的角膜内皮细胞移植研究新进展

随着组织细胞培养方法的不断完善,大量研究表明,人类及灵长目动物的角膜内皮细胞(corneal endothelial cell,CEC)可以在组织培养的条件下生长并形成单层,其细胞形态与活体人CEC类似,这为采用培养的CEC进行移植以恢复损伤的内皮功能提供了技术支持。本文就有关CEC体外培养、载体选择以及移植方法的最新进展进行综述,对目前仍需解决的问题和前景进行展望。     

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。     

角膜内皮细胞载体培养及移植的研究进展

自七十年代末Gospodarowicz等^[1]首次将培养的牛角膜内皮(corneal endothelium，CE)细胞种植于异体角膜片载体上行同种异体CE细胞移植后，已进行了大量有关CE细胞载体培养及移植的实验^[2～5]。本文拟就其研究进展作一综述。     

组织工程角膜内皮细胞移植的研究与进展

由于角膜供体材料严重短缺以及供体年龄限制,穿透性角膜移植术的临床广泛开展受到严重制约,应用组织工程体外培养高密度、具备常规六角形态和健康内皮功能的角膜内皮细胞是当前研究的热点。本文就角膜内皮种子细胞的来源、载体的选择及角膜内皮细胞移植方法、免疫机制等方面研究的最新进展进行综述,总结目前研究面临的问题并展望其前景。     

数字微脉冲超声乳化术后角膜内皮的变化

目的　观察应用微脉冲技术进行晶状体超声乳化吸出术对角膜内皮细胞的损害。方法　 2 0 0 2年 10月～ 12月老年性白内障 46例 (5 3眼 )进行微脉冲超声乳化手术 ,于术前及术后 3月测量角膜中央内皮细胞密度及六边形细胞比率 ,计算内皮丢失率。结果　术前术后内皮细胞密度分别为 (2 .474± 2 .74) /mm2 和 (2 2. 18± 2 .66) /mm2 (P <0 . 0 1) ,平均细胞丢失率为9. 7%,六边形细胞比率术前术后无明显改变。结论　采用微脉冲技术进行超乳手术 ,对角膜内皮细胞的损害较轻 ,细胞形态无明显改变。     

选择性角膜内皮移植术

长期以来,治疗角膜内皮病变标准的方法为穿透性角膜移植术(PK)。虽然总体上说,PK手术成功率高,但由于术后视力恢复时间长,且可出现多种并发症,如高度散光／不规则散光、植片排斥反应以及与缝线和切口愈合相关的并发症等,因此并非一种理想的手术方式。过去十几年内,出现了多种选择性角膜内皮移植术,如经角膜瓣途径的内皮移植术、经角巩膜袋途径的内皮移植术及带活性内皮细胞的Descemet’s膜移植术等。与PK手术相比,这些新的手术方法理论：有着多方面的优势,初步的临床结果亦令人鼓舞,有可能成为替代传统PK手术治疗角膜内皮病变的更有效的方法。     

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。     

后弹力层撕除角膜内皮移植术研究现状

后弹力层撕除角膜内皮移植术对角膜表面形态和屈光影响小,视力恢复好,主要治疗各种原因导致的角膜内皮失代偿,如:Fuchs角膜内皮营养不良、大泡性角膜病变、虹膜-角膜内皮综合征或穿透性角膜移植失败。本文就后弹力层撕除角膜内皮移植术技术改进和临床结果的新进展作一综述     

Corneal endothelial status in the subtypes of primary angle closure glaucoma

Purpose: To study the corneal endothelium and pachy­metry in eyes with different subtypes of primary angle closure glaucoma (PACG), as compared to controls. Methods: A cross‐sectional study was conducted on 30 consecutive patients in each subtype of PACG, subacute, acute and chronic, and 30 age and refraction matched controls. The parameters recorded included gonioscopy, optic disc evaluation, applanation tonometry, specular microscopy and central ultrasonic pachymetry. Results: The mean endothelial cell counts in the four groups were as follows: subacute PACG 2396 ± 271 cells/mm², acute PACG 1597 ± 653 cells/mm², chronic PACG 2229 ± 655 cells/mm² and controls 2461 ± 321 cells/mm². The mean endothelial cell count in the fellow eyes of subacute PACG, acute PACG and chronic PACG patients was 2294 ± 305 cells/mm², 2388 ± 226 cells/mm² and 2108 ± 203 cells/mm², respectively (NS). The acute PACG patients had significantly lower endothelial cell counts (P < 0.001) as compared to the other three groups. Eyes in which the acute attack of angle closure persisted for less than 72 h had a mean endothelial cell count of 2016 ± 306 cells/mm², as compared to 759 ± 94.4 cells/mm² in eyes with an attack lasting for 72 h or more (P < 0.001). The endothelial count was also significantly lower in eyes with chronic PACG as compared to control eyes (P < 0.001). There was increased pleomorphism and polymegathism of the corneal endothelial cells seen in eyes with resolved acute and chronic PACG. The mean central corneal thickness was 531.4 ± 25.3 µm in eyes with subacute PACG, 567.9 ± 37.3 µm in eyes with acute PACG, 526.4 ± 31.9 µm in eyes with chronic PACG and 525 ± 12.6 µm in control eyes. The acute PACG eyes had a significantly higher corneal thickness (P < 0.001) when compared to all the other groups. Conclusion: There is a significant decrease in the corneal endothelial cell density in eyes that have had an acute attack of angle closure glaucoma and in eyes with chronic PACG. The endothelial cell population in eyes with sub­acute PACG and in the fellow eyes of all subtypes of PACG is not significantly different from the normal population.     

Corneal endothelial response to induced myopia in the chicken

Purpose : To investigate the role of corneal endothelial surface enlargement in the chicken myopia model in inducing corneal endothelial changes. Methods : Lid suture was performed on one eye of 1‐day‐old cockerels. Five chickens were killed at 1 week, and four chickens killed at each of 3 weeks, 6 weeks, and 10 weeks postnatal. The endothelial morphology was obtained by flat mounting the endothelial surface and the subsequent digitisation. Comparisons were undertaken between the control unsutured eye and the lid‐sutured eye endothelium, and between the central endothelial areas compared to the peripheral endothelial areas in both the myopic and the normal corneas. Calculation of the contribution to the endothelial change by hypertrophy and mitosis were calculated using Bahn’s formula. Results : Total endothelial surface area increased significantly over time in the myopic model compared to control eyes but the mean cell area of endothelial cells remained the same for both the enlarged myopic endothelial surface area and in the normal controls. Sampling from the central and the peripheral corneal endothelial surface also disclosed no difference. The mean cell area did increase steadily with age but was the same for both normal and myopic corneas. Conclusions : It would appear that there are equal contributions from hypertrophy and mitosis in the myopic group and the normal corneal group with a slightly increasing trend towards mitotic activity in the myopic corneal endothelial layer.     

Prospects for endothelial transplantation

BACKGROUND: The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells (HCEC), transplantation of these cells may be an alternative therapeutic option. MATERIALS AND METHODS: In this review methods for the in vitro cultivation of HCEC and their transplantation onto the Descemet membrane of donor corneas are described. RESULTS: In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors. Dependent on the culture conditions, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behaviour. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation onto donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation status of the cells in vitro. Highest cell numbers up to 3000 cells/mm2 were achieved using a SV40-transformed HCEC-cell line. Monolayer integrity could be demonstrated by positive staining for integrins and light junction proteins, and pump function of the newly established endothelium was proven by perfusion studies. CONCLUSIONS: Methods to transplant HCEC onto human denuded corneas have been successfully established to reconstruct human corneas. Recent developments in genetic manipulation of cells and tissue engineering will be of great help in constructing suitable corneas for keratoplasty. Thus corneal endothelial cell transplantation is one of the promising future possibilities to provide corneas of high quality for patients. Furthermore, improvement of the transplantation technique may lead to a method to directly manipulate the diseased endothelium of patients with corneal endothelial dystrophies.     

人脐静脉血管内皮细胞在体移植替代猴角膜内皮细胞的形态学分析

目的　使用去除后弹力层的恒河猴自体角膜为细胞载体,将培养的人脐静脉血管内皮细胞（ｈｕｍａｎｕｍｂｉｌｉｃａｌｖｅｉｎｅｎ-ｄｏｔｈｅｌｉａｌｃｅｌｌｓ,ＨＵＶＥＣ）种植到角膜内表面,观察ＨＵＶＥＣ替代恒河猴角膜内皮细胞的功能情况以及ＨＵＶＥＣ在恒河猴眼内生长的情况。方法　取恒河猴６只随机分为３组:实验组（３只）、实验对照组（２只）、空白对照组（１只）。实验组:用离心沉淀法将培养的ＨＵＶＥＣ移植到去除后弹力层的恒河猴自体角膜内表面,之后将自体角膜缝回植床;实验对照组:将撕除部分后弹力层的术眼角膜植片原位缝回植床;空白对照组:取下术眼角膜植片不做任何处理原位缝回植床。术后观察各组角膜植片透明情况;实验组及实验对照组于术后３０ｄ及６０ｄ、空白对照组于术后６０ｄ行术眼摘除,标本做病理切片、ＣＤ３４免疫组化及扫描电镜,观察房角结构及ＨＵＶＥＣ在角膜植片内表面形态分布。结果　实验组角膜植片维持了一定的厚度和透明性,而实验对照组角膜植片发生严重大泡性改变。病理切片示实验组角膜内表面可见一层细胞生长,ＣＤ３４染色阳性,提示为血管内皮细胞;实验对照组角膜内表面未见任何细胞生长;空白对照组角膜植片保留完整后弹力层及内皮细胞层。扫描电镜示实验组有ＨＵＶＥＣ单层在角膜内表面生长但有大量白细胞聚集及少量细胞碎片嵌顿于小梁网;实验对照组角膜内表面残留胶原纤维样物质,无细胞生长;空白对照组见完整六边形角膜内皮细胞层。结论　ＨＵＶＥＣ能够在撕除后弹力层的恒河猴角膜内表面生长并发挥一定的屏障作用,维持角膜的厚度和透明性,但会产生较重的排斥反应。