体外培养重建角膜组织的技术探讨

目的：探讨体外重建角膜组织的三维培养技术。方法：原代培养角膜上皮,基质和内皮细胞,利用硫酸软骨素,透明质酸钠,I型胶原蛋白和微孔材料,建立三维培养系统,体外重建角膜组织结构,观察非醛交联,醛交联和微孔材料等三类人工角膜的透明度,常规病理组织学检测细胞在三维培养系统中生长状态。结果：非醛交联类重建人工角膜质地较软,透明度较低,光镜可见纤维结构,醛交联类重建人工角膜质地较韧,透明性较高,光镜可见均质结构,非吸收微孔材料类细胞长人较少,较难体外重建人工角膜,结论：利用生物工程技术能够体外重建人工角膜,非醛交联类结构较规则,醛交联类透明度较高。     

应用三维培养技术体外重建角膜组织的初步研究

目的 探讨三维培养技术体外重建角膜组织的理论基础及临床应用价值。方法 原代培养人角膜上皮、基质及内皮细胞、通过胶原凝胶三维培养系统,体外重建角膜结构,应用裂隙灯检测人工角膜的透明度,采用常规病理组织学及透射电镜观察细胞在三维培养系统中生长的特性。结果 重建的角膜组织为良好的三层结构;角膜上皮细胞形成复层结构;内皮细胞形成单层结构,且排列规则;角膜基质细胞呈散在分布。电镜观察、细胞在Ⅰ型胶原中存活并合成胶原纤维。结论 利用组织工程技术,能够体外重建结构较规则,透明度较高的角膜组织。     

组织工程技术体外重建角膜上皮层的实验研究

目的 利用组织工程技术体外重建角膜上皮组织，为眼表重建提供良好的移植材料及方法。方法 以去除上皮细胞的羊膜基底膜为载体，分别种植兔角膜缘组织块及原代培养扩增的角膜缘上皮细胞，体外培养重建角膜上皮层，并进行形态、组织学、超微结构观察及免疫细胞化学检测。结果 角膜缘上皮细胞在羊膜上粘附生长并增殖，体外培养10天左右即形成3～4层上皮细胞，复合角膜上皮组织由羊膜基底膜和复层上皮细胞组成，细胞表达特异性角蛋白CK3，与生理状态下的角膜上皮组织相近。结论 以羊膜为载体培养角膜缘上皮细胞可体外重建组织工程化角膜上皮组织，作为眼表重建移植材料良好的来源。     

角膜缘干细胞在重建眼表中的作用研究进展

正常角膜上邓小平的完整性依赖于基底层角膜缘干细胞的不断增殖。角膜缘于细胞对角膜上皮的再生起着重要的作用。如果角膜缘干细胞因各种原因严重缺乏，会导致持续性角膜上皮糜烂、角膜新生血管和假性翼状胬肉形成，角膜的完整性和透明性将受到破坏。本对角膜缘干细胞的性质、特点、分布、检测、各种干细胞移植术、干细胞的体外培养及其适宜的载体等进行综述，以便对角膜缘干细胞进行更深入的研究。     

人表皮生长因子对体外培养兔角膜内皮细胞影响的实验研究

目的：明确人表皮生长因子（ｈｕｍａｎ Ｅｐｉｄｅｒｍａｌ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ,ｈＥＧＦ）对角膜内皮细胞的影响。方法：采用体外培养的兔角膜内皮细胞,观察接种后不同时点ｈＥＧＦ对其生长状态的影响;兔角膜片内皮损伤模型,离体培养后行Ｈ－Ｅ染色和Ｈ＾３－ＴｄＲ掺入放射自显影,观察ｈＥＧＦ对其修复的影响。结果：兔角膜内皮细胞体外培养ｈＥＧＦ组细胞数高于对照组,并使细胞形态产生纺锤形改变;角膜内皮细胞     

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。     

不同体外方法培养兔角膜缘上皮细胞作用的研究

自从1986年Schermer等发现角膜缘干细胞位于角膜缘基底Vogt栅栏内后，对角膜缘干细胞缺失和角膜上皮病变治疗有了许多新的疗法，其中以角膜缘上皮细胞体外培养法治疗上述疾病比较有效。目前，多数学者采用组织块法进行角膜缘上皮细胞体外培养，但在培养系中干细胞能否从组织块中迁移出来尚有争议。另外，最近角膜缘上皮单细胞悬浮液超低温冷藏、复苏以及体外培养技术越来越受到人们的关注，成为角膜缘干细胞研究中的一项重要的课题。此种培养方法既可以将角膜缘干细胞和其他角膜上皮细胞长期保存在超低温状态下，减少细胞传代培养次数，[第一段]     

人角膜缘干细胞在体外不同载体上培养的实验研究

目的 寻找人角膜缘干细胞体外培养的载体。方法 人角膜缘干细胞原代培养单细胞层传代到羊膜、卵壳膜、聚乳酸膜、藻酸盐凝胶4种载体和6种培养板中,倒置显微镜观察、记录细胞生长情况。对6孔板传代细胞行生物特性检测,对载体上培养细胞行组织学检测。结果 6孔板中传代细胞对AE－5,4G10．3表达阳性。羊膜、卵壳膜和聚乳酸膜上有细胞生长,7天左右形成单层;藻酸盐凝胶载体上未见细胞生长。结论 传代细胞具有角膜缘干细胞生物特性;羊膜、卵壳膜和聚乳酸膜适合角膜缘干细胞传代培养。     

兔角膜缘干细胞的体外培养

目的 探寻兔角膜缘干细胞体外培养方法。方法 分别用20％小牛血清营养液和20％胎牛血清营养液兔角膜缘干细胞行体外培养，观察细胞贴壁生长情况；同时对生长良好的原代细胞进行消化传代，进一步观察传代细胞的形态和生长情况。结果 20％胎牛血清营养液组，角膜缘干细胞在培养48－72h有80％贴壁生长，7－10天形成良好单层，细胞呈圆形、卵圆形或多边形，类似角膜上皮细胞；传代培养细胞形态仍正常，生长良好，但混有成纤维细胞。20％小牛血清营养液培养72h，仅有20％细胞贴壁生长，且细胞形态极不规则，有细胞“拉网”现象，培养14天仍未形成单层。结论 角膜缘干细胞的培养较常规细胞培养难，需要特殊培养基。20％胎牛血清营养液能作为角膜缘干细胞培养的基础液。     

角膜内皮细胞体外培养的研究进展

角膜内皮是角膜的重要组成部分,由单层的角膜内皮细胞(corneal endothelial cell,CEC)组成,在维持角膜正常的透明度方面发挥着重要的作用.CEC在出生后即丧失增殖能力,一旦受损或丢失会导致不可逆性角膜疾病.但是,大量的研究表明CEC可以在体外增殖,这为体外培养人CEC用于角膜疾病的治疗提供了可行性,但至今仍没有建立一套标准的CEC体外培养方法.本文将归纳总结近年来CEC体外培养方法.以利于今后优化CEC体外培养体系.     

人角膜上皮细胞体外培养的研究进展

角膜上皮细胞位于角膜外表面,人角膜上皮细胞体外培养有助于充分了解角膜细胞的生物学特性、构建体外疾病模型以及更好地发挥其临床作用.我们就近年来关于人角膜上皮细胞体外培养提取部位,提取方法,培养基、细胞因子与底物以及鉴别方法研究进展进行综述.     

双氢青蒿素对体外培养兔角膜上皮细胞毒性作用的实验研究

目的 评价双氢青蒿素对体外培养的兔角膜上皮细胞的毒性作用。设计 实验研究。研究对象 体外培养兔角膜上皮细胞。方法 体外培养兔角膜上皮细胞,加入不同稀释浓度（1.6%、0.8%、0.4%、0.2%、0.1%、0.05%和0.025%）的双氢青蒿素共同孵育,同时以洗必泰和聚六亚甲基双胍（PHMB）做对照药物。在培养后2、6、24小时,光学显微镜下观察细胞形态,LIVE/DEAD染色观察药物作用后兔角膜上皮细胞的活性改变。主要指标 细胞形态改变。结果 培养后2、6小时,0.4%双氢青蒿素作用后的细胞出现圆化,细胞间隙加宽;0.0125%洗必泰作用后的细胞开始出现细胞间隙加宽,边界不清;0.003%PHMB作用后的细胞开始出现细胞边界不清,细胞内颗粒样改变。培养后24小时,0.1%的双氢青蒿素作用的角膜上皮细胞尚存在活性,0.025%的双氢青蒿素对角膜上皮细胞无明显影响。0.006%洗必泰和0.003%PHMB可导致角膜上皮细胞的死亡。结论 双氢青蒿素对体外培养的兔角膜上皮细胞的毒性较低,其对活体角膜细胞的作用尚待进一步研究。     

体外培养中牛角膜内皮细胞和虹膜色素上皮细胞的

目的明确FasL在体外培养的角膜内皮细胞和虹膜色素上皮细胞（IPE）中FasL的表达情况及其意义。材料和方法采用酶辅助的显微分离法分离新生牛眼IPE细胞,酶消化法分离角膜内皮细胞,进行体外细胞培养。抗FasL抗体、S-P试剂盒（DAB显色）对传一代IPE细胞和角膜内皮细胞进行免疫组化染色。结果角膜内皮细胞FasL染色呈阳性;IPE细胞及对照组均为阴性。结论在前房免疫赦免的形成中,角膜内皮细胞主要通过表达和分泌FasL发挥作用,而IPE细胞则主要通过表达和分泌有免疫抑制作用的细胞因子以及通过一种非Fas-FasL诱导细胞凋亡的通路来阻止T细胞活化、增殖。     

体外培养中牛角膜内皮细胞和虹膜色素上皮细胞的FasL表达

目的：明确FasL在体外培养的角膜内皮细胞和虹膜色素上皮细胞（IPE）中FasL的表达情况及其意义,材料和方法：采用酶辅助的显微分离法分离新生牛眼IPE细胞,酶消化法分离角膜内皮细胞,进行体外细胞培养,抗FasL染色呈阳性;IPE细胞及对照组均为阴性。结论：在前房免疫赦免的形成中,角膜内皮细胞主要通过表达和分泌FasL发挥作用,而PE细胞则主要通过表达和分泌有免疫抑制作用的细胞因子以及通过一种非FasL－FasL诱导细胞凋亡的通路来阻止T细胞活化、增殖。     

Mitotic activity of rabbit corneal epithelium in vitro

It is difficult to maintain corneal epithelial cells for several passages in culture. At a first step to establish a culture system for corneal epithelium, the authors evaluated factors which seemed to be involved in the growth kinetics and mitotic activity of rabbit corneal epithelium in vitro. First of all, to compare the growth kinetics of peripheral and central epithelium, we measured the areas and distances of the epithelium that outgrew from peripheral and central explants in culture. The peripheral corneal epithelium grew 1.2-to 1.6-fold better than the central corneal epithelium. However, there was no significant difference between them. Secondly, we evaluated the effects of epidermal growth factor (EGF), cholera toxin (CTX) and insulin, which were contained in supplemented hormonal epithelial medium (SHEM) developed by Jumblatt et al, on the mitotic activity of rabbit corneal epithelium. Among these drugs, only EGF had the ability to increase the epithelial cell number after 10 days of incubation. By contrast, CTX which elevated the level of cyclic AMP inhibited the proliferation of the epithelium.     

Biology in vitro of corneal epithelium and endothelium

Four main areas were explored: 1) the proper medium for culturing corneal tissue; 2) the effect of serum on tissue growth in vitro; 3) the interrelationships in vitro between corneal epithelium and endothelium; and 4) the biology of cultures of whole corneas (organ cultures). Modified Eagle's minimal essential medium (MEM) proved to be an excellent culture fluid. Corneal tissue could be grown in MEM without serum or clot, thus providing a defined culture medium. The biology in vitro of outgrowths of multilayered corneal epithelium and monolayered corneal endothelium are discussed. Contact inhibition between epithelium and endothelium is demonstrated in whole corneal (organ) cultures.From the Laboratory of Ophthalmic Pathology, Scheie Eye Institute, and the Departments of Ophthalmology and Pathology, University of Pennsylvania Medical School.This study was supported by the National Institutes of Health, Training Grant T01-EV-0070-03 from the National Eye Institute.Candidate's thesis for membership in the American Ophthalmological Society, Accepted by the Committee on Thesis May 1975.     

Toxic effects of ophthalmic preservatives on cultured rabbit corneal epithelium

We investigated the effects of the ophthalmic preservatives thimerosal and sorbic acid on the proliferation and survival of rabbit corneal epithelial cells in tissue culture. Normally, explants of corneal epithelium grow vigorously during the first 7 days in culture. With 0.004% thimerosal present in the culture medium, the normal proliferation of corneal cells is suppressed completely. When 0.1% sorbic acid is present, proliferation is delayed and the lifespan of the corneal cells is reduced. After a 1-h exposure to concentrations of thimerosal of 0.0005% or greater, virtually all corneal cells present in established cultures are killed. These results suggest that use of ophthalmic preparations containing these chemicals may affect the metabolic and proliferative capacity of the corneal epithelium adversely.     

An in vitro model of leukocyte mediated injury to the corneal epithelium

To enhance efforts directed at unraveling the role and mechanisms of leukocytes in mediating injury to corneal epithelium, an isolated bovine corneal cup was developed and evaluated. Bovine peripheral leukocytes and lysates were added to the corneal epithelial surface of isolated cornea for various periods after which the degree of morphologic changes and cell damage were assessed using light and electron microscopy. Results of these studies indicate that leukocyte/epithelial cell interactions are characterized by five successive stages: (1) leukocyte adhesion to superficial layer of the epithelium, (2) leukocyte penetration beneath the superficial epithelium, (3) epithelial cell injury, (4) leukocyte phagocytosis of killed epithelial cells and (5) ulceration and total destruction of the full thickness of the epithelial layer. The above sequence appears to be both time and dose dependent; that is epithelial cells exposed to leukocytes for short periods (5-60 minutes) or to low dose levels (10(5) - 10(7) cells/ml) shows leukocyte adhesion and penetration beneath the superficial layer of the epithelium, (stage 1 and 2), while longer exposures (2-3 hours) or higher numbers of leukocytes (10(7) - 10(8) cells/ml), leads to deeper penetration of epithelium by leukocytes and epithelial injury (Stages 3,4,and 5). We also observed that direct contact of intact leukocytes with epithelial cells is apparently necessary to induce this type of injury. These findings demonstrate the ability of leukocytes to destroy corneal epithelial cells and the value of this new ocular model for studies of the basic immunology of ocular inflammation.