Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice

PURPOSE: Severe ocular surface diseases and injuries cause loss of the corneal limbal epithelium, leading to re-epithelialization by bulbar conjunctival cells, resulting in vascularization of the cornea, conjunctival scarring, and loss of visual acuity. In this study, the optimal culture condition for induction of differentiation of epithelial progenitor cells from embryonic stem (ES) cells was determined for use in transplantation to damaged cornea in mice. METHODS: Mouse ES cells were cultured on Petri dishes coated with several extracellular matrix proteins, and the markers for epithelial cells were analyzed with RT-PCR and Western blot analysis. The optimal condition for induction of epithelial progenitor cells was determined, and the progenitors were transplanted onto mouse eyes with corneal epithelia that had been damaged by exposure to n-heptanol. RESULTS: Epithelial progenitors were successfully induced by culturing mouse ES cells on type IV collagen for 8 days. These progenitors expressed keratin (K)12, which is specific to corneal epithelial cells, and cell surface CD44 and E-cadherin, both of which are essential in corneal epithelial wound healing. Complete re-epithelialization of the corneal surface occurred within 24 hours after transplantation. The resultant corneal epithelial cells expressed markers of the grafted cells, and no teratomata were observed during the follow-up period. CONCLUSIONS: Epithelial progenitors were successfully induced in vitro from ES cells and were applicable as grafts for treating corneal epithelial injury. ES cells may become an unlimited donor source of corneal epithelial cells for corneal transplantation and may restore useful vision in patients with a deficiency of limbal epithelial cells. This is an important first trial toward assessing the use of ES cells to reconstruct corneal epithelial cells.     

种植人骨髓间充质干细胞的猪角膜板层移植治疗兔角膜损伤的初步研究

目的研究种植人骨髓间充质干细胞（MSCs）的猪角膜基质治疗兔角膜损伤的可能性。方法用全骨髓贴壁法分离纯化人MSCs并传代,流式细胞仪检测免疫表型及诱导成脂、成骨分化鉴定。12只新西兰白兔随机分为2组,实验组取第3代MSCs接种于去上皮的猪角膜基质上,培养4 d后移植到广泛损伤的兔角膜上,对照组单纯移植去上皮猪角膜基质。术后2、4、8周,取各实验眼行组织学检查,观察移植的MSCs及猪角膜基质的存活、转归及移植局部的反应。免疫组织化学、免疫荧光染色检测移植后角膜上皮细胞角蛋白12的表达。结果培养获得的MSCs中CD29阳性者占95.97%,CD44阳性者占96.49%,CD90阳性者占92.79%,CD105阳性者占94.66%,CD34阳性者占0.59%,CD45阳性者占0.36%,符合MCSs的免疫表型,并可以诱导成脂及成骨分化。实验组MSCs接种到去上皮猪角膜基质后贴附、生长迅速,术后植片在植床上存活良好,无排斥反应,角膜较对照组透明,新生血管少,而对照组在移植后发生排斥反应。实验组角膜免疫组织化学及免疫荧光染色均检测出CK12阳性细胞。结论种植MSCs的猪角膜基质移植到损伤兔角膜后可以存活,MSCs可以分化为角膜上皮样细胞,具有构建组织工程角膜的潜能。     

诱导人脐带间充质干细胞分化为角膜上皮样细胞的研究

背景眼表疾病导致的角膜盲已成为全球致盲性角膜疾病中的主要原因之一。随着组织工程技术的发展和进步,组织工程角膜为眼表疾病的治疗开辟了新的途径。目的观察体外培养的人脐带间充质干细胞（UC—MSCs）移植到兔角膜基质后的分化发育情况,探讨人UC-MSCs分化为角膜上皮细胞以及治疗兔角膜损伤的可行性。方法获取人脐带组织,采用Ⅳ型胶原酶消化法分离纯化人UC—MSCs并传代,取第3代细胞用于扩增和实验。流式细胞仪检测细胞的免疫表型及诱导成骨分化鉴定。24只新西兰大白兔按随机数字表法随机分为2个组,将人UC—MSCs接种于去上皮的猪角膜基质上,培养4d后行实验组兔左眼板层角膜移植;对照组以相同的手术方法单纯移植去上皮猪角膜基质。术后对角膜定期行活体激光共焦显微镜检查,并分别于术后2、4、8周摘除各组实验眼行组织病理学和免疫荧光检查,评价移植到兔角膜基质的人UC-MSCs的存活、分化以及移植局部的反应等;应用免疫荧光技术检测移植后角膜上皮细胞中角蛋白3（CK3）、CKl2以及转运蛋白G超家族成员（ABCG2）的表达。结果消化培养的人UC—MSCs呈圆形,细胞胞体较大,贴壁后细胞呈长梭形。培养获得的人UC—MSCs的细胞表型CD105^＋／CD29^＋／CD44^＋／CD34^-／CD45^-,并可诱导分化为成骨细胞。实验组人ISC—MSCs接种到去上皮猪角膜基质后贴附良好、生长迅速,术后植片在植床上存活良好,种植了人UC-MSCs的去上皮猪角膜移植到兔眼,可见实验组受体角膜较对照组透明,未见明显新生血管,在活体共焦显微镜下可见新生的角膜上皮样细胞,未发生免疫排斥反应。免疫荧光检测可见在重建的角膜上皮层检测到CK3及CKl2的阳性表达,而未见ABCG2的表达。结论将种植了人UC—MSCs的猪角膜基质移植到损伤的兔角膜后,人UC—MSCs可以存活、增生并分化为角膜上皮样细胞,可用于修复甚至重建损伤的角膜表层。     

Transplantation of human embryonic stem cells onto a partially wounded human cornea in vitro

Purpose: The aim of this study was to investigate whether cells originating from human embryonic stem cells (hESCs) could be successfully transplanted onto a partially wounded human cornea. A second aim was to study the ability of the transplanted cells to differentiate into corneal epithelial‐like cells. Methods: Spontaneously, differentiated hESCs were transplanted onto a human corneal button (without limbus) with the epithelial layer partially removed. The cells were cultured on Bowman’s membrane for up to 9 days, and the culture dynamics documented in a time‐lapse system. As the transplanted cells originated from a genetically engineered hESC line, they all expressed green fluorescent protein, which facilitated their identification during the culture experiments, tissue preparation and analysis. To detect any differentiation into human corneal epithelial‐like cells, we analysed the transplanted cells by immunohistochemistry using antibodies specific for CK3, CK15 and PAX6. Results: The transplanted cells established and expanded on Bowman’s membrane, forming a 1–4 cell layer surrounded by host corneal epithelial cells. Expression of the corneal marker PAX6 appeared 3 days after transplantation, and after 6 days, the cells were expressing both PAX6 and CK3. Conclusion: This shows that it is possible to transplant cells originating from hESCs onto Bowman’s membrane with the epithelial layer partially removed and to get these cells to establish, grow and differentiate into corneal epithelial‐like cells in vitro.     

Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea

AIM: To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS: A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy (TEM) and hematoxylin and eosin (H&E) staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype. RESULTS: The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process. In vitro, the methyl thiazolyl tetrazolium results revealed that extracts of the acellular ostrich corneas (AOCs) had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes. The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea (APC) transplantation. The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer. CONCLUSION: We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.     

骨髓间充质干细胞移植治疗眼表损害的初步实验研究

目的观察体外培养的人骨髓间充质干细胞（hMSCs）移植到碱烧伤的兔角膜表面后,干细胞的成活、迁移和分化情况。方法采用NaOH溶液制作兔角膜碱烧伤模型,1个月后将培养有hMSCs的羊膜缝合到碱烧伤的兔角膜表面,以羊膜作为对照组,在裂隙灯显微镜下观察角膜的临床改变。术后1个月,摘除眼球,石蜡切片,苏木素-伊红（HE）染色观察角膜组织结构的变化,并进行抗人核抗体和细胞角蛋白12（CK12）的免疫组化染色以观察hMSCs的分布和分化情况。结果兔眼碱烧伤1个月后,角膜表面和基质层均可见大量血管,角膜呈瓷白色混浊,表面粗糙干燥,出现大量杯状细胞。hMSCs移植1个月后,角膜表面粗糙程度减轻,新生血管略有减少,但是角膜混浊未见明显改善,角膜表面杯状细胞消失;角膜表面和基质浅层存在抗人核抗体染色阳性的细胞;角膜表面细胞CK12染色阳性,而基质层未见CK12阳性细胞。对照组在羊膜移植1个月后,角膜状况较移植前无明显改善,角膜表面仍可见杯状细胞,角膜各层均未见抗人核抗体和CK12染色阳性的细胞。结论hMSCs移植到碱烧伤兔角膜表面后,能够成活并向角膜基质迁移,未发生移植排斥反应。hMSCs由于所在部位不同,可以在周围组织的诱导下向不同方向发生分化,角膜表面的细胞向角膜上皮细胞分化。而基质层中的细胞未分化为角膜上皮细胞。移植后角膜表面结膜化程度有所减轻。     

人角膜及角膜缘上皮细胞分化标记的检测

目的检测分化标记在人角膜及角膜缘上皮细胞的表达,以了解角膜及角膜缘细胞分化状态,旨在发现新的角膜上皮干细胞的阴性标记。方法获取人角膜及角膜缘组织,对冰冻切片及整个角膜组织行免疫荧光染色检测分化标记钙粘连素E、角蛋白3(CK3)、角蛋白12(CK12)、缝隙连接蛋白43、巢蛋白(nestin)和包壳蛋白(involucrin)的表达,经荧光显微镜及激光扫描共焦电镜观察,并行半定量RT-PCR以检测其相关分化标记基因的表达。结果分化标记CK3、CK12、缝隙连接蛋白43、巢蛋白和包壳蛋白在角膜和角膜缘上皮的表层细胞表达,角膜缘基底细胞不表达。激光扫描共焦电镜观察及RT-PCR结果显示角膜缘基底上皮细胞不表达细胞CK3、连接蛋白43和巢蛋白,而角膜上皮细胞则明显表达。结论角膜及角膜缘表层上皮较为成熟分化,而角膜缘基底细胞具有未分化细胞的特征,很可能是干细胞的部位。     

自体骨髓间充质干细胞移植改善重度眼表损伤临床观察

目的 评价自体骨髓间充质干细胞在严重眼表损伤移植后的治疗效果.方法 选取了化学烧伤4例,Stevens-Johnson综合征2例.骨髓间充质干细胞取自患者,细胞培养14 d后,经细胞检测鉴定为骨髓间充质干细胞,将细胞种植在羊膜表面,细胞扩增融合达90％后使用.手术切除表面结膜化组织达角膜缘外3 mm,将羊膜上培养的自体骨髓间充质干细胞移植到角膜及角膜缘表面,术后予抗生素、激素、人工泪液点眼治疗;病人定期随访,进行裂隙灯显微镜及印迹细胞学检查.结果 6例患眼均覆盖了含有自体骨髓间充质干细胞的羊膜片,术后1周角膜上皮完整.3例患者视力有改善,2例患者视力保持术前水平,1例患者视力下降.术后所有患者眼表面光滑程度有所改善,但新生血管没有明显减少.术后12周印迹细胞学检查结果,1例患者角膜表面可检测到上皮样细胞及杯状细胞.结论 自体骨髓间充质干细胞移植可以改善眼表条件,有潜在应用于治疗角膜缘功能障碍的价值.     

异体角膜缘上皮细胞培养后移植的实验研究

目的观察含培养的角膜缘上皮细胞的羊膜片移植到角膜缘干细胞损伤的异体兔眼角膜上的效果。方法把羊膜为载体组织块培养的角膜缘上皮细胞移植到角膜缘干细胞损伤的异体兔眼角膜上,术后观察角膜混浊度、角膜新生血管、角膜上皮等情况,定期兔进行病理组织学检查。结果角膜缘上皮细胞在羊膜上培养16d形成3～4层,用含有培养的异体角膜上皮细胞的羊膜片移植的12只兔眼,术后第5天,损伤的角膜表面全部上皮化,第16天开始,12只兔眼逐渐出现排斥反应。结论含培养的角膜缘上皮细胞羊膜片异体移植术后早期角膜全部上皮化,晚期则出现排斥反应。     

Experimental transplantation of cultured human limbal and amniotic epithelial cells onto the corneal surface.

PURPOSE: Tissue-cultured corneal epithelial transplantation is a novel procedure that uses tissue-cultured epithelial cells to restore severely damaged ocular surfaces. In this study, we used tissue-cultured human limbal and amniotic epithelial cells as donor cells to investigate the feasibility of this procedure for reestablishment of a damaged ocular surface in experimental conditions. METHODS: Primary human limbal epithelial cultures were established from banked limbal tissue. Amniotic epithelial cells were isolated from serologically screened human placenta and maintained in a specialized nutrient medium. Suspended cells (5 x 10(5)/ml) were seeded onto the concave surface of collagen corneal shields and incubated at 37 degrees C for 2-3 days. These cell-covered shields were then placed on a denuded stromal surface in organ culture and on New Zealand albino rabbit ocular surfaces that had the native epithelium previously removed. Specimens were collected 24, 48, 72, and 96 h later from organ-cultured corneal buttons and recipient animals, processed, and evaluated histologically. RESULTS: The cells grown on the collagen shield were spread uniformly and unpolarized after 48 h in culture. They were repolarized and tightly adhered to the recipient corneal stroma 24 h after transplantation, as demonstrated by formation of cell-substrate hemidesmosomes (HDs) and donor-specific antigen immunostaining. The donor cells were retained in six of 15 rabbits receiving limbal cells and four of 12 rabbits receiving amniotic cells for as long as 10 days after surgery. CONCLUSION: Cultured human limbal and amniotic epithelial cells can be successfully transplanted onto a denuded corneal surface where they adhere tightly to underlying stroma by hemidesmosomes.     

Transplantation of cultivated autologous oral mucosal epithelial cells in patients with severe ocular surface disorders

BACKGROUND/AIMS: To determine outcomes of transplants of cultivated autologous oral epithelial cells in patients with severe ocular surface disorders. METHODS: The eyes (n = 6) of four patients with Stevens-Johnson syndrome (three eyes) or chemical burns (three eyes) were studied. Autologous oral epithelial cells, grown for 2-3 weeks on a denuded amniotic membrane carrier in the presence of 3T3 fibroblasts, were air lifted. The resultant sheet was transplanted onto the damaged eye, and acceptance of the sheet by the corneal surface was confirmed 48 hours after surgery. The success of ocular surface reconstruction, graft survival, changes in visual acuity, and postoperative complications were assessed and the quality of the cultivated oral epithelial sheet was evaluated histologically. RESULTS: At 48 hours after transplant, the entire corneal surface of all six eyes was free of epithelial defects indicating complete survival of the transplanted oral epithelium. Visual acuity was improved in all eyes. During follow up (mean 13.8 (SD 2.9) months), the corneal surface remained stable, although all eyes manifested mild peripheral neovascularisation. CONCLUSIONS: Autologous oral epithelial cells grown on denuded amniotic membrane can be transplanted to treat severe ocular surface disorders.     

Successful primary culture and autologous transplantation of corneal limbal epithelial cells from minimal biopsy for unilateral severe ocular surface disease

BACKGROUND: Patients with severe unilateral ocular surface disease require reconstruction of the damaged ocular surface. We succeeded in culturing primary corneal limbal epithelial cells taken from minimal biopsy and, once grown, transplanting them on denuded amniotic membrane (AM). METHODS: Autologous corneal limbal epithelial cells from a 3 mm(2) biopsy of the uninjured eye were grown for 3 weeks on a denuded AM carrier. The resultant sheet was then transplanted onto the unilateral severely chemically injured eye. RESULTS: Minimal biopsy showed the autologous cultivated corneal epithelial cells to have 4-5 layers of sufficient stratification and to be well differentiated. At 19 months post-transplantation, the ocular surface epithelium was stable and there were no epithelial defects. CONCLUSION: We document that it is possible to produce sufficiently stratified, well differentiated, autologous cultivated corneal limbal epithelium on AM from a minimal biopsy of the donor eye and to transplant it onto the injured eye.     

以异种角膜脱细胞基质为载体构建角膜上皮-载体-内皮复合物的实验研究

目的探讨以异种角膜脱细胞基质为载体,体外构建生物角膜的可能性和方法。方法应用去垢剂1％TritonX-100冷冻干燥处理制备猪角膜脱细胞基质载体,在其上皮面和内皮面分别接种兔角膜上皮细胞和内皮细胞,体外培养2周。将复合物制成组织切片,在光镜下观察组织形态（HE染色）,采用免疫组织化学方法检测角膜上皮特异性细胞角蛋白3（CK3）,使用锥虫蓝联合茜素红染色观察内皮细胞,在扫描电镜下观察上皮面和内皮面的超微结构。结果体外培养生物角膜获得上皮、无细胞基质和内皮三层复合结构。4或5层复层上皮中以扁平细胞为主,胞质内特异性CK3表达阳性;内皮层为一连续的单层扁平细胞,细胞活性良好,锥虫蓝联合茜素红染色显示组织呈典型的蜂巢样镶嵌结构。扫描电镜下观察,在载体的上皮面细胞呈复层样生长,形态近似扁平与梭形之间;内皮面为多边形单层细胞,表面具有微绒毛结构。结论构建的生物角膜为上皮一脱细胞基质载体一内皮复合物,初具角膜的雏形结构。异种角膜脱细胞基质提供了良好的细胞生长界面,有望成为体外构建角膜的载体材料。     

Localization of keratin in the cells of the cornea in aphakia and normal mouse embryos

Abnormal lens morphogenesis in the aphakia mutant in the mouse often results in a club-shaped elongated 'lens' that remains attached to the surface epithelium by a persistent connecting stalk, which is partially solid and partially cystic. Usually, the cells are continuous with the surface epithelium of the cornea and also with the cuboidal cells lining the corneal inner surface. Immunofluorescence with keratin antiserum not only gave positive reactions with the corneal epithelial cells, but also with many cells of the lens stalk, including its cysts, and with islands of cells on the inside of the cornea. These keratin-containing 'endothelial' cells may be the product of metaplasia of the endothelium into epithelium-like cells. Alternatively, they may also be the result of abnormal migration of epithelial cells into the eye or of abnormal differentiation of neural crest cells.     

人角膜上皮干细胞的识别

目的 探讨人角膜上皮干细胞的分子标记。方法 对人角膜和角膜缘部位行组织学检查以分析角膜缘解剖结构。对人角膜切片和整个角膜组织行免疫组织化学染色以检测中央角膜和角膜缘部位未分化标记,如核蛋白p63、乳腺癌抵抗蛋白（ABCG2,BCRP1）、烯醇化酶α、整合素拍、胡及β1、表皮生长因子受体（EGFR）、细胞角蛋白19（CK19）、14（CK14）及转铁蛋白受体（CDT1）的表达,经荧光显微镜和激光扫描共焦显微镜观察。对角膜中部和角膜缘上皮细胞的mRNA进行半定量逆转录聚合酶链反应（RT—PCR）和原位杂交以检测其相关基因的表达。结果 角膜缘部位横向切片显示角膜缘上皮细胞为乳头放射状排列,对应于Vogt栅栏环境。未分化标记整合素β1、EGFR、烯醇化酶α及CK19在角膜缘基底细胞胞质染色较表层细胞更强;p63、ABCG2、整合素胡蛋白仅见于角膜缘基底部上皮细胞。激光扫描共焦显微镜观察和RT—PCR结果显示角膜缘表达p63、ABCG2、整合素胡蛋白及mRNA。原位杂交显示p63仅表达于角膜缘基底层细胞。结论 角膜缘上皮呈乳头放射状排列,角膜缘干细胞群具有复合标记：p63表达于细胞核、ABCG2表达于胞质、整合素胡表达于胞膜。采用这些标记复合体,可将角膜缘干细胞群与其他上皮细胞区分。     

A case of bilateral corneal epithelial dysplasia characterized by laser confocal biomicroscopy and cytokeratin immunofluorescence

PURPOSE: To describe a case of bilateral corneal epithelial dysplasia in which each lesion was characterized by both laser confocal biomicroscopy and cytokeratin immunofluorescence. METHODS: A 52-year-old Japanese woman with bilateral corneal epithelial dysplasia was treated by corneal epithelial debridement. We observed the affected area with laser confocal biomicroscopy before and after treatment and examined the immunofluorescence of cytokeratins to examine the characteristics of the abnormal epithelial cells. RESULTS: Laser confocal biomicroscopy revealed the atypical epithelial cells in all layers of the corneal epithelium as well as the reconstituted normal structure of the corneal epithelium after epithelial debridement. Immunofluorescence of cytokeratin 12 (K12) and K4 revealed the presence of four types of cells (those positive for one, both or neither of these cytokeratins) in each lesion. CONCLUSION: Cells expressing both K12 and K4 probably represented dysplastic cells that had invaded the cornea via the limbus and adopted characteristics of corneal epithelial cells. Cells lacking both K12 and K4 were probably either undifferentiated cells or epithelial cells in which cytokeratin expression had not been initiated.     

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。     

培养的兔羊膜上皮细胞构建角膜表层移植片的研究

目的应用培养的兔羊膜上皮细胞（AECs）体外构建复层上皮细胞-角膜基质移植材料,探讨利用AECs重建角膜表层的可行性。方法取妊娠晚期新西兰大白兔（27～28孕周）的羊膜,制成AECs单细胞悬液,用含血清和表皮生长因子（EGF）的DMEM／F12培养液培养、传代,利用免疫组织化学单克隆抗体AE1／AE3、AE5检测培养的AECs中细胞角蛋白ck3／12的表达;将体外培养的2～3代兔AECs种植在新鲜兔角膜基质上,利用气-液界面培养法使之复层化,体外构建复层上皮细胞-角膜基质移植材料,进行光学显微镜和扫描电镜观察,并进行免疫组织化学测定。结果体外培养的兔AECs呈现单克隆抗体AE1／AE3、AE5表达阳性,AECs在新鲜兔角膜基质上能形成形态类似于正常角膜上皮细胞的3～5层复层结构,且复层化后的上皮细胞单克隆抗体AE5表达阳性。结论应用培养的AECs能成功构建类似角膜表层的复层上皮细胞-角膜基质移植材料,AECs可能成为重建角膜表层的一种新的细胞来源。     

Development of a biopolymeric keratoprosthetic material. Evaluation in vitro and in vivo

Based on the results of in vitro and in vivo experiments, we have determined that the optimal material for the central transparent portion of a perforating keratoprosthesis is a polyvinyl alcohol copolymer hydrogel. The material supports the maintenance and growth of corneal epithelium in vitro as shown by population doublings and transmission electron microscopy. Discs preseeded with epithelial cells were cultured in vitro and transplanted into rabbit corneas. The proliferation of these cells in vivo was demonstrated using 3H-thymidine. Other experiments showed that the preseeded cells not only migrated from the central disc onto the peripheral rim of the host cornea but also that host peripheral epithelial cells migrated onto the anterior surface of the disc. The experiments described in this paper demonstrate that corneal epithelial cells preseeded onto hydrogel discs and transplanted into rabbit corneas remain adherent and are capable of proliferating.     

Ex vivo transfer of Smad7 decreases damage to the corneal endothelium after penetrating keratoplasty

PURPOSE: To determine whether transient gene transfer and expression of the intracellular antagonist of transforming growth factor beta (TGF-beta), Smad7, to corneal endothelial cells decreases corneal endothelial cell damage after penetrating keratoplasty in a rabbit model. METHODS: Rabbit corneas were transfected ex vivo with replication-deficient adenoviruses encoding Flagtagged Smad7, Flag-tagged Smad3, or LacZ (termed AdCMV-Smad7, AdCMV-Smad3, AdCMV-LacZ) and then transplanted to normal rabbits. Expression of the exogenous Smads and phosphorylation of endogenous Smad2 in the transplanted corneal endothelium were examined by immunoblotting and immunohistochemistry with anti-Flag or anti-phosphorylated Smad2 antibodies. Cellular density and morphological changes in the corneal endothelium of the transplanted cornea were evaluated by scanning electron microscopy after transplantation of the Smad-transfected corneas. RESULTS: Transplanted AdCMV-Smad7-transfected corneas significantly inhibited the decrease in cellular density and accelerated wound healing at the host-graft junction when compared with transplanted AdCMV-LacZ-transfected corneas. Transplanted AdCMV-Smad3-transfected corneas showed decreased cellular density and delayed wound healing at the host-graft junction. CONCLUSIONS: Ex vivo gene transfer of Smad7 to corneal endothelial cells inhibits the decrease in cellular density and accelerates wound healing after penetrating keratoplasty in rabbits. Thus, modulation of Smad7 expression in corneal endothelial cells may decrease corneal endothelial cell damage after penetrating keratoplasty.