A narrative review of limbal stem cell deficiency&severe ocular surface disease

Background and Objective:Limbal stem cell deficiency(LSCD)describes the clinical condition when there is dysfunction of the corneal epithelial stem/progenitor cells and the inability to sustain the normal homeostasis of the corneal epithelium.The limbal stem cells are located in a specialized area of the eye called the palisades of Vogt(POV).There have been significant advances in the diagnosis and management of LSCD over the past decade and this review focuses on the pathophysiology of LSCD,its clinical manifestations,diagnosis,and causes.Methods:Papers regarding LSCD were searched using PubMed to identify the current state of diagnosis and causes of LSCD published through to June 2022.Key Content and Findings:LSCD is clinically demonstrated by a whorl-epitheliopathy,loss of the POV,and conjunctivalization of the cornea.The diagnosis of this condition is based on clinical examination and aided by the use of impression cytology,in vivo confocal microscopy,and anterior segment optical coherence tomography(asOCT).There are many causes of LSCD,but those which are most common include chemical injuries,aniridia,contact lens wear,and Stevens-Johnson syndrome(SJS).Conclusions:While this condition is most commonly encountered by corneal specialists,it is important that other ophthalmologists recognize the possibility of LSCD as it may arise in other co-morbid eye conditions.

Focus on limbal stem cell deficiency and limbal cell transplantation

Limbal stem cell deficiency(LSCD)causes severe vision impairment and can lead to blindness,representing one of the most challenging ocular surface disorders.Stem cell deficiency can be congenital or,more often,acquired.The categorization of ocular surface transplantation techniques is crucial to achieving treatment homogeneity and quality of care,according to the anatomic source of the tissue being transplanted,genetic source,autologous or allogenic transplantation(to reflect histocompatibility in the latter group),and cell culture and tissue engineering techniques.The aim of this minireview is to provide a summary of the management of LSCD,from clinical characteristics and therapeutic outcomes to the development of novel therapeutic approaches.The manuscript also briefly summarizes recent findings in the current literature and outlines the future challenges to overcome in the management of the major types of ocular surface failure.

Cell-based therapies for limbal stem cell deficiency: a literature review

Background and Objective:Limbal stem cell deficiency(LSCD)is characterized by the insufficiency of limbal stem cells to maintain the corneal epithelium.Severe cases of LSCD may be treated with limbal transplantation from healthy autologous or allogeneic limbal tissue.Multiple cell-based therapies have been studied as alternative treatments to improve success rates and minimize immunosuppressive regimens after allogeneic transplants.In this review,we describe the success rates,and complications of different cell-based therapies for LSCD.We also discuss each therapy’s relative strengths and weaknesses,their history in animal and human studies,and their effectiveness compared to traditional transplants.Methods:PubMed was searched for publications using the terms LSCD,cell-based therapy,cultivated limbal epithelial transplantation(CLET),cultivated oral mucosal epithelial transplantation(COMET),and mesenchymal stem cells from 1989 to August 2022.Inclusion criteria were English language articles.Exclusion criteria were non-English language articles.Key Content and Findings:current cell-based therapies for LSCD are CLET and non-limbal epithelial cells.Non-limbal epithelial cell methods include COMET,conjunctival epithelial autografts,and mesenchymal stem/stromal cells(MSCs).Moreover,several alternative potential sources of non-limbal cells have described,including induced pluripotent stem cells(iPSCs),human embryonic stem cells(hESCs),human dental pulp stem cells,hair follicle bulge-derived epithelial stem cells,amniotic membrane epithelial cells,and human umbilical cord lining epithelial cells.Conclusions:Cell-based therapies are a promising treatment modality for LSCD.While CLET is currently the only approved cell-based therapy and is only approved in the European Union,more novel methods have also been shown to be effective in human or animal studies thus far.Non-limbal epithelial cells such as COMET are also an alternative treatment to allogeneic transplants especially as a surface stabilizing procedure.iPSCs are currently being studied in early phase trials and have the potential to revolutionize the way LSCD is treated.Lastly,cell-based therapies for restoring the limbal niche such as mesenchymal stem cells have also shown promising results in the first human proof-of-concept study.Several potential sources of non-limbal cells are under investigation.

Midterm outcomes of penetrating keratoplasty following allogeneic cultivated limbal epithelial transplantation in patients with bilateral limbal stem cell deficiency

AIM:To evaluate the midterm outcomes of penetrating keratoplasty(PK)following allogeneic cultivated limbal epithelial transplantation(CLET)for bilateral total limbal stem cell deficiency(LSCD).METHODS:Ten patients(10 eyes)with bilateral LSCD were enrolled in this prospective noncomparative case series study.Each participant underwent PK approximately 6 mo after a CLET.Topical tacrolimus,topical and systemic steroids,and oral ciclosporin were administered postoperatively.Best-corrected visual acuity(BCVA),intraocular pressure(IOP),ocular surface grading scores(OSS),corneal graft epithelial rehabilitation,persistent epithelial defect(PED),immunological rejection,and graft survival rate were assessed.RESULTS:The time interval between PK and allogeneic CLET was 6.90±1.29(6-10)mo.BCVA improved from 2.46±0.32 log MAR preoperatively to 0.77±0.55 log MAR post-PK(P<0.001).Kaplan-Meier analysis of mean graft survival revealed graft survival rates of 100%at 12 and 24 mo and 80.0%at 36 mo.PEDs appeared in 5 eyes at different periods post-PK,and graft rejection occurred in 4 eyes.The total OSS decreased from 12.4±4.4 before allogeneic CLET to 1.4±1.51 after PK.CONCLUSION:A sequential therapy design of PK following allogeneic CLET can maintain a stable ocular surface with improved BCVA despite the relatively high graft rejection rate.

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.

Therapeutic effect of secretome from TNF-αstimulated mesenchymal stem cells in an experimental model of corneal limbal stem cell deficiency

AIM:To explore the secretome efficacy in tumor necrosis factor(TNF)-αstimulated mouse mesenchymal stem cells(MSCs)in a murine model of corneal limbal alkali injury.METHODS:Corneal limbal stem cell deficiency(LSCD)was created in the eyes of male C57 mice.Concentrated conditioned medium from TNF-αstimulated MSCs(MSC-CMT)was applied topically for 4 wk,with basal medium and conditioned medium from MSCs as controls.Corneal opacification,corneal inflammatory response,and corneal neovascularization(NV)were evaluated.Corneal epithelial cell apoptosis,corneal conjunctivation,and inflammatory cell infiltration were assessed with TUNEL staining,CK3 and Muc-5 AC immunostaining,and CD11 b immunofluorescence staining,respectively.The effect of TSG-6 was further evaluated by knockdown with short hairpin RNA(sh RNA).RESULTS:Compared to the controls,topical administration of MSC-CMT significantly ameliorated the clinical symptoms of alkali-induced LSCD,with restrained corneal NV,reduced corneal epithelial cell apoptosis,and inhibition of corneal conjunctivation.In addition,MSC-CMT treatment significantly reduced CD11 b+inflammatory cell infiltration,and inhibited the expression of pro-inflammatory cytokines(IL-1β,TNF-αand IL-6).Furthermore,the promotion of corneal epithelial reconstruction by MSC-CMT was largely abolished by TSG-6 knockdown.CONCLUSION:Our study provides evidence that MSCCMT enhances the alleviation of corneal alkali injuries,partially through TSG-6-mediated anti-inflammatory protective mechanisms.MSC-CMT may serve as a potential strategy for treating corneal disorders.

Quantification of corneal neovascularization after ex vivo limbal epithelial stem cell therapy

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization.METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animalfree culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization,vessel caliber(VC), and invasive area(IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity(BCVA),epithelial transparency, and impression cytology were also measured.RESULTS: One year after surgery, successful cases showed a reduction(improvement) of all three parameters of corneal neovascularization [neovascular area(NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31%(P =0.035), invasion area29.37%(P =0.018) and VC 14.29%(P =0.072). BCVA improved in all eyes(mean follow-up, 76 ±21mo).Epithelial transparency improved significantly from 2.00 ±0.93 to 0.88±1.25(P =0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up.CONCLUSION: This method of analyzing andmonitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method,successful cases could be differentiated from failed cases.

Limbal epithelial stem cells in corneal surface reconstruction

Cornea serves as the partial front barrier and major light reflection organ of the eye.The integrity of corneal surface is essential for ocular function.Injuries or congenital diseases could significantly destruct the homeostasis of the ocular surface,especially the microenvironment of limbal epithelial stem cells(LESCs),and will eventually cause dysfunction of corneal regeneration and diminish of LESCs.The loss of LESCs by different reasons are named limbal stem cell deficiency(LSCD),which is one of the leading cause of vision loss worldwide.To restore the corneal surface,LESC transplantation in the form of tissue or cell cultures is currently a viable and promising method to treat LSCD.In this review,we aim to introduce the characters and niche of LESCs,and discuss different aspects of its application in cornea surface reconstruction.

Induced pluripotent stem cells as a potential therapeutic source for corneal epithelial stem cells

Corneal blindness caused by limbal stem cell deficiency(LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered by the shortage of donors. Pluripotent stem cell technology including embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs) have opened new avenues for treating this disease. iPSCs-derived corneal epithelial cells provide an autologous and unlimited source of cells for the treatment of LSCD. On the other hand, iPSCs of LSCD patients can be used for iPSCs-corneal disease model and new drug discovery. However, prior to clinical trial, the efficacy and safety of these cells in patients with LSCD should be proved. Here we focused on the current status of iPSCs-derived corneal epithelial cells used for cell therapy as well as for corneal disease modeling. The challenges and potential of iPSCs-derived corneal epithelial cells as a choice for clinical treatment in corneal disease were also discussed.

Gene therapy and genome editing for primary immunodeficiency diseases

In past two decades the gene therapy using genetic modified autologous hematopoietic stem cells(HSCs)transduced with the viral vector has become a promising alternative option for treating primary immunodeficiency diseases(PIDs).Despite of some pitfalls at early stage clinical trials,the field of gene therapy has advanced significantly in the last decade with improvements in viral vector safety,preparatory regime for manufacturing high quality virus,automated CD34 cell purification.Hence,the overall outcome from the clinical trials for the different PIDs has been very encouraging.In addition to the viral vector based gene therapy,the recent fast moving forward developments in genome editing using engineered nucleases in HSCs has provided a new promising platform for the treatment of PIDs.This review provides an overall outcome and progress in gene therapy clinical trials for SCID-X,ADA-SCID,WAS,X-CGD,and the recent developments in genome editing technology applied in HSCs for developing potential therapy,particular in the key studies for PIDs.

角膜缘微环境细胞的分离培养研究进展

角膜缘微环境细胞(LNCs)是位于角膜缘、与角膜缘上皮干细胞(LESCs)紧密相连的一类间充质干细胞,能同时表达间充质标志物和多种胚胎干细胞标志物,对调节LESCs的静息、自我更新以及分化状态起着重要作用。近年研究表明,LNCs可通过胶原酶消化法、上皮块消化法、中性蛋白酶-胶原酶消化法以及组织块培养法进行体外分离培养,通过3D Matrigel共培养、Transwell共培养可研究LNCs与LESCs之间的相互作用关系。LNCs与LESCs可通过SDF-1/CXCR4、Notch、BMP、Wnt、Sonic Hedgehog、KIT/AKT等多种信号通路以及神经生长因子、角质形成细胞因子、胰岛素样生长因子等多种细胞因子相互作用。LNCs现已成为角膜上皮组织工程、眼表重建以及角膜再生等研究中的一大热点。本文就LNCs的研究背景、体外分离培养方法、与LESCs的相互作用机制及其应用前景等进行综述。

Axenfeld-Reiger syndrome: A search for the missing links

BACKGROUND Axenfeld-Rieger syndrome(ARS)is a rare cause of congenital glaucoma and may result in loss of vision.ARS is mostly autosomal dominant in nature characterized by developmental abnormalities in the angle of anterior chamber and iris of the eye,also associated with structural abnormalities in the body.AIM To study and observe the demographics and clinical findings in a very rare ocular disease known as ARS.METHODS Case records of symptomatic patients attending Ophthalmology outpatient department and diagnosed to have ocular hypertension or glaucoma in 3 years from March 2017 to March 2020 were evaluated to search for cases diagnosed with ARS.Records of all patients diagnosed with ARS were then analysed for demographic and clinical characterization as well as management and success of therapy.RESULTS Eight out of ten patients with positive clinical signs were symptomatic and had glaucoma.One of these patients had limbal stem cell deficiency and another had vernal keratoconjunctivitis.CONCLUSION Clinical characterization of ARS is important for making a definitive diagnosis and determining prognosis.

角膜缘干细胞缺乏动物模型建立方法研究进展

角膜缘干细胞缺乏(LSCD)是眼科常见疾病,严重的LSCD是角膜病致盲的主要原因之一。LSCD新疗法的探索仍是当今研究的热点。建立LSCD动物模型有助于更深入了解LSCD的发病机制以及评估治疗方法的有效性。目前,LSCD建模的主要方式有机械法、灼烧法、化学法、化学+手术法及基因敲减法。机械法成熟可靠,应用广泛,但可能会引起严重的机械损伤。其中,使用旋转毛刺工具造模损伤均匀且创伤小,但对于工具的使用需要一定的经验积累。灼烧法造模表型稳定,但操作复杂可控性差。化学法造模快速简单,病因与临床病例的吻合度较高,但损伤的深度及范围不可控。化学+手术法角膜表面光滑平整,但易导致角膜中央与角膜缘厚度不一致,影响新生血管生长。基因敲减法复杂且动物饲养困难,应用较少。虽然造模方法众多,但各研究中的造模质量参差不齐。因此,建立可靠、成功率高的LSCD动物模型十分必要。本文就近年来常用LSCD动物模型的建立方法及优缺点进行综述,以期为后续构建合适模型提供借鉴。

口腔黏膜上皮培养与眼表移植的研究进展

人角膜上皮细胞由角膜缘干细胞不断补充,从而维持眼表稳态及正常视觉功能。热化学烧伤、Stevens-Johnson综合征等眼表损伤或疾病可导致角膜缘干细胞缺损,严重影响患者视力。当双眼角膜缘干细胞缺损时可考虑非角膜缘性自体上皮组织移植。口腔黏膜上皮细胞是眼表重建的重要种子细胞来源,体外培养构建的细胞片已在临床应用中取得良好效果。本文围绕载体、培养条件及技术等影响口腔黏膜上皮片培养的各种因素,以及细胞片应用于临床眼表移植的优缺点,对利用口腔黏膜上皮治疗角膜缘干细胞缺损的研究进展进行综述,以期为安全有效地重建眼表上皮提供研究方向。

人胚胎干细胞来源角膜上皮样细胞用于治疗兔角膜缘干细胞缺乏

目的 通过小分子化合物诱导人胚胎干细胞(human embryonic stem cells, hESCs)分化为角膜上皮样细胞,观察角膜上皮样细胞治疗兔角膜缘干细胞缺乏(limbal stem cell deficiency, LSCD)的效果。方法 利用拟胚体联合小分子化合物方法,将hESCs H9细胞系向角膜上皮样细胞分化,将角膜上皮样细胞在去上皮羊膜片上培养构建重组细胞膜片。选取造模成功的12只LSCD兔,按照随机数表法分为对照组和实验组,对照组接受去上皮的的羊膜片治疗,实验组接受角膜上皮样细胞膜片移植治疗。术后通过裂隙灯、前节照相等方法,对角膜透明度、新生血管、荧光素钠染色情况进行评分,并于移植后4周观察角膜病理切片结构。结果 成功将hESCs诱导为角膜上皮样细胞,细胞形状和结构类似角膜上皮细胞。病理结果显示,实验组的角膜形成复层上皮结构,上皮下炎症细胞浸润轻,治疗效果明显优于对照组。进行细胞移植治疗后发现,实验组的角膜透明度高、新生血管少,角膜评分明显优于对照组(P<0.05)。诱导后的角膜上皮样细胞的角膜上皮标记物ΔNP63和CK12的表达与hESCs H9相比差异具有统计学意义(P<0.05)。结论 采用拟胚体联合小分子化合物可以成功诱导出角膜上皮样细胞,角膜上皮样细胞膜片移植到兔体内后可有效治疗LSCD。

组织工程口腔黏膜上皮治疗角膜缘干细胞缺陷症的实验研究

目的探讨体外培养的自体组织工程口腔黏膜上皮重建兔角膜上皮的可行性。方法制作兔角膜缘干细胞缺陷模型32只,实验组Ⅰ～Ⅲ以自体口腔黏膜上皮细胞为种子细胞制作组织工程上皮,移植到实验组模型兔角膜表面,分别观察2周、1个月、3个月,对照组移植空白载体膜,观察3个月。术后裂隙灯显微镜下观察,以角膜新生血管、混浊度及上皮染色评分评价移植效果。用组织病理、免疫组织化学和印迹细胞技术评价角膜上皮重建的可能性。结果模型兔角膜混浊,有大量新生血管和杯状细胞。实验组移植后角膜透明,印迹细胞检查PAS(-)。实验组与对照组术后角膜总评分差异有统计学意义(P=0.000),p63表达阳性,角膜上皮的组织特点及角蛋白的表达与正常角膜上皮相似。结论组织工程口腔黏膜上皮在角膜基质微环境的诱导下可分化为角膜样上皮细胞,有重建角膜上皮的作用。

兔全角膜缘干细胞缺乏模型的构建和鉴定

背景角膜缘干细胞缺乏（LSCD）可导致各种眼表功能异常，严重的LSCD会导致角膜结膜化、慢性炎症、持续的角膜上皮缺失、角膜混浊和伴发的长期视力丧失。目的研究成功构建兔全LSCD模型的适宜方法和最佳时间，观察造模后各时间点模型眼角膜的病理结构改变。方法选择3～5月龄健康日本大耳白兔20只，用手术刀切除左眼360。角膜缘内1mm、角膜缘外2mm的角膜缘上皮组织和剩余的全部中央角膜上皮组织和浅层基质，切除组织厚度为100～150μm。分别在术后2～5周行裂隙灯检查，以角膜混浊、角膜新生血管情况和上皮荧光素钠染色情况为观察指标，按照国际通用LSCD模型评分标准对模型眼进行评分。分别于术后2、3、4、5周获取模型眼的角膜组织标本，行苏木精一伊红染色和过碘酸希夫染色观察角膜组织结构和杯状细胞的改变，用间接免疫荧光染色法检测细胞角蛋白3（CK3）在角膜组织中的表达。手术后不同时间点造模成功率的差异比较采用Fisher精确概率法。结果术后第2、3、4、5周时的模型成功率分别为12．5％、62．5％、81．3％和87．5％，术后第3周造模成功率明显高于第2周，差异有统计学意义（P=0．009）；第3周与第4周相比差异无统计学意义（P=0．465），第3周与第5周相比差异有统计学意义（P=0．049），第4周与第5周相比差异无统计学意义（P=0．200）。造模成功的平均时间为（3．21±0．80）周。造模成功的标准为模型眼角膜明显混浊，角膜新生血管形成和荧光素钠染色阳性。模型眼的组织病理学检查示角膜基质水肿，较多炎性细胞浸润和新生血管形成。过碘酸希夫染色发现，仅在造模后第5周的角膜组织中发现杯状细胞。各时间点造模成功模型眼角膜组织中均未见到CK3阳性表达。结论用手术切除角膜缘及全部中央角膜上皮的方法可以成功构建全LSCD动物模型，造模成功的时间约为手术后3．2周。

家兔角膜缘干细胞缺失病理模型的制作

根据角膜缘干细胞定位于角膜缘上皮基底层,为角膜上皮增殖分化的源泉并保持角膜上皮完整性的理论。将20只家兔随机分为4组,每组5只,分别将角膜缘上皮全周或半周板层手术切除,或用1mol/LNaOH直接擦除等方法破坏角膜缘干细胞,探索制作角膜缘干细胞完全缺失病理模型的有效途径。结果表明,处理后4周,全周角膜缘上皮板层手术切除,中央角膜上皮层用1mol/LNaOH擦除的5只试验家兔角膜表面全部血管化、结膜化,未发生睑球粘连,角膜基质胶原纤维完整未见溃疡、穿孔等病变,细胞印迹学检查为结膜表型,可作为实验性角膜缘干细胞移植的病理模型;全周角膜缘上皮板层手术切除,中央角膜上皮用生理盐水擦除的5只试验家兔,有2只为结膜表型,另3只为角膜表型,观察期内结果不稳定;半周角膜缘上皮板层手术切除,中央角膜上皮层用生理盐水擦除的5只试验家兔,角膜表面透明,全部为角膜表型;直接用1mol/LNaOH擦除角膜缘和中央角膜上皮的试验家兔,有4只角膜基质胶原纤维断裂、溶解,并伴有严重的溃疡、穿孔、睑球粘连等病变,不能用于移植试验,另1只角膜表面透明,未见结膜和新生血管长入,细胞印迹学检查为角膜表型。

角膜上皮重建的研究进展

角膜上皮的增生与更新依赖于角膜缘干细胞,角膜缘干细胞缺乏可导致视力障碍。组织工程化角膜上皮的研究是眼科研究的一个热点。我们就近年来在组织工程角膜上皮重建中应用的非眼来源干细胞的新进展作一简要综述。

角膜缘上皮细胞移植重建眼表的实验研究

目的探讨角膜缘上皮细胞构建的角膜上皮植片重建眼表的可行性和有效性。方法采用碱烧伤法建立完全性角膜缘干细胞缺陷的SD大鼠动物模型,以羊膜为载体培养人角膜缘上皮细胞,移植重建角膜缘干细胞缺陷眼表,观察术后眼表状态的改变,RT-PCR和免疫组化检测术后10、20、30、45 d及60 d角膜组织CK3/12和P63的表达。结果体外培养的人角膜缘上皮细胞移植术后,完全性角膜缘干细胞缺陷眼表状态较术前和对照组都有不同程度改善;免疫组化和RT-PCR检测表明体外培养角膜缘上皮细胞移植术后角膜组织60 d内P63和CK3/12表达无明显改变。结论以去上皮羊膜为载体,体外培养人角膜缘上皮细胞构建的角膜上皮植片能够重建完全性角膜缘干细胞缺陷眼表。