角膜缘干细胞移植治疗角膜疾病的研究进展

角膜缘干细胞在维持正常角膜上皮完整性以及角膜损伤修复中有重要作用.角膜缘干细胞损伤所致的角膜缘干细胞缺失可导致严重的角膜病变.目前治疗角膜缘干细胞缺失的主要方法是细胞移植治疗.本文就角膜缘干细胞的体外培养、鉴定、移植方法以及新的细胞来源等问题进行综述.     

角膜缘干细胞培养体系的研究进展

正常角膜上皮形态稳定和功能的维持是依靠角膜缘干细胞的不断增殖和移行来实现。近年来，角膜缘干细胞的重要作用逐渐被人们所认识。本文从角膜缘干细胞的定位、体外取材、分离培养、培养条件的选择、常用体外培养的载体、鉴别方法等对角膜缘干细胞培养体系进行综述。     

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。     

组织工程化角膜上皮的新方法:在对温度敏感的细胞培养面上进行角膜缘干细胞的培养及移植

角膜缘干细胞严重缺损患者可行角膜缘移植治疗,但供体角膜组织有限。众多学者尝试在多种不同载体上体外培养和扩增角膜缘干细胞构建角膜上皮移植片并进行移植。然而,载体的选择可影响患者的预后,而且生物材料引起的感染也在所难免。为此,作者介绍一种新的不需载体也不需缝合的角膜缘干细胞培养和移植方法。     

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]     

培养兔自体角膜缘干细胞移植的实验研究

目的 观察以羊膜为载体的兔角膜缘于细胞膜片移植治疗兔角膜缘于细胞缺损的效果。方法 制造兔角膜缘干细胞缺损的动物模型,以右眼为实验眼,从兔左眼取角膜缘组织,将兔角膜缘干细胞消化下来,接种于铺有羊膜的无菌六孔培养板中,待细胞形成多层角膜上皮细胞后,对兔角膜缘干细胞缺损动物模型行角膜缘干细胞羊膜移植术,并对治疗后的角膜进行裂隙灯及病理学检查。结果 临床和组织病理学染色证实：体外培养的兔角膜缘干细胞可在羊膜上继续增生、分化为多层角膜上皮细胞;角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮完整、基质细胞浸润减轻、新生血管减少或消失。结论 应用角膜缘干细胞羊膜移植术可恢复其角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘的细胞屏障功能。     

组织工程化角膜上皮的新方法：在对温度敏感的细胞培养面上进行角膜缘干细胞的培养及移植[英]／Nishida K…∥Transplantation．

角膜缘干细胞严重缺损患者可行角膜缘移植治疗，但供体角膜组织有限。众多学者尝试在多种不同载体上体外培养和扩增角膜缘干细胞构建角膜上皮移植片并进行移植。然而，载体的选择可影响患者的预后，而且生物材料引起的感染也在所难免。为此，作者介绍一种新的不需载体也不需缝合的角膜缘干细胞培养和移植方法。     

角膜缘干细胞在重建眼表中的作用研究进展

正常角膜上皮的完整性依赖于基底层角膜缘干细胞的不断增殖。角膜缘干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。本文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。     

培养角膜缘干细胞羊膜移植治疗碱烧伤动物的实验研究

目的 观察培养生长于羊膜的角膜缘干细胞移植的治疗角膜缘碱烧伤伤的效果。方法 将兔角膜缘干细胞在的代培养后接种于羊膜,对新西兰大白兔角膜缘碱烧伤动物模型行角膜缘干细胞羊膜移植术,并对治疗后的角膜进行临床及病理学检查。结果 体外培养的兔角膜缘士细胞可在羊膜上继续增殖、分化为密集的角膜上皮细胞层;角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮完整基质细胞浸润减轻、新生血管减少。组织病理学染色证实,角膜缘     

角膜缘龛的稳态维持及损伤重建

人角膜上皮的完整性由角膜缘上皮干细胞的不断更新补充来维持,而角膜缘的特殊区域—角膜缘龛,是干细胞赖以生存的微环境。角膜缘龛由多种细胞、信号分子以及细胞外基质构成,各成分的协调作用维持了角膜缘龛的稳态,并促进了角膜缘干细胞的增生、迁移及分化。先天性、外伤性、免疫性疾病等任何对角膜缘龛稳态产生干扰的因素均可导致干细胞的功能异常。对于角膜缘干细胞缺乏症患者,通过细胞外基质的再生、生物活性因子及间充质干细胞的应用等策略来重建角膜缘龛,对于角膜缘干细胞的恢复及长期维持具有重要意义。(国际眼科纵览,2020,44:391-396)     

组织块联合胰酶消化法培养兔角膜缘干细胞的初步探索

目的：建立一种简便、高效的兔角膜缘干细胞原代培养方法。

方法：获取兔角膜缘组织,采用组织块联合胰蛋白酶消化法进行兔角膜缘干细胞体外培养,倒置显微镜下观察细胞生长情况,HE染色观察细胞形态和结构特点,并运用免疫组织化学技术进行细胞鉴定。

结果：采用组织块联合胰蛋白酶消化法可以简便、快速地培养出兔角膜缘干细胞,显微镜下动态观察细胞生长良好,有较高的增殖能力; HE染色证实细胞形态和结构正常; AE5及P63免疫组织化学鉴定呈阳性。

结论：采用组织块联合胰蛋白酶消化共同培养的方法,建立了一种简便、高效的兔角膜缘干细胞原代培养模式。     

骨髓间充质干细胞在角膜损伤修复中的应用和研究进展

骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)可在体内外培养诱导分化为角膜上皮细胞、角膜缘上皮细胞和角膜基质细胞等。BMSC移植后可分化为角膜上皮细胞,有效修复损伤的角膜上皮细胞组织,能表达多种细胞因子,减少炎症细胞浸润,减轻细胞损伤,并抑制细胞凋亡,降低角膜移植后的排斥反应; BMSC还能分化促进角膜缘干细胞增殖。也可在载体上利用BMSC构建生物角膜,进行眼表修复,此为基础研究提供了新的研究方向,为临床治疗角膜损伤类疾病提出了新的临床治疗思路,在基础研究和临床运用方面都有非常巨大的潜力和广阔的前景。本文就BMSC在角膜损伤修复中的研究现状和进展进行综述。     

自体角膜缘干细胞移植联合MMC对翼状胬肉疗效的Meta分析

目的:探讨自体角膜缘干细胞移植术联合丝裂霉素(MMC)治疗翼状胬肉对术后复发及角膜上皮修复时间的影响。方法:计算机检索Cochrane Datebase,Pubmed,中国期刊全文数据库、中国知网、维普中文科技期刊数据库、万方数据库。检索时限从建库到2013-02。纳入比较自体角膜缘干细胞移植联合MMC与自体角膜缘干细胞移植治疗翼状胬肉对胬肉复发及角膜上皮修复时间影响的随机对照试验(RCT)。数据统计分析采用Cochrane协作网提供的RevMan5.1软件。结果:共初检出217篇文章,最终纳入12个RCT,共1044眼。对12项RCT进行Meta分析显示:自体角膜缘干细胞移植术联合MMC与单纯自体角膜缘移植术比较,术后复发率有统计学意义(OR=0.23,95%CI:0.15～0.36,Z=6.40,P<0.01);对于其中研究到术后角膜上皮修复时间的6项RCT进行Meta分析显示:自体角膜缘干细胞移植术联合MMC与单纯自体角膜缘移植术比较,术后角膜上皮修复时间无统计学意义(MD=0.62,95%CI:0.62～1.85,Z=0.98,P=0.33)。结论:自体角膜缘干细胞移植术中运用MMC可明显减少术后复发,对角膜上皮修复的时间无明显影响。     

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 animal-free 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 area 29.37% (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 and monitoring 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.     

Improvement in the method of cultivated corneal epithelial sheet generation

Stromal cells and/or soluble factors such as growth factors have been considered until now as the main niche of corneal epithelial stem cells. We found that cultivated epithelial cells had epithelial clusters when cultured, maintaining the sheet structure. Therefore, we hypothesized that the epithelial cells themselves might be a niche of the stem cells, and the cell clusters could be maintained during the gentle enzymatic digestion of the limbal epithelial sheets. The main methods of generating cultivated limbal epithelial sheet for clinical application are, the cell suspension method (complete enzymatic digestion of limbal epithelia), and the explant method (cells grown from limbal explants). For cell suspension, it is good to expand cells and generating sheets quickly, but it is not clear wheather these sheets maintain the normal homeostatic levels of the progenitor cells. Explants maintain homeostatic levels more similar to in vivo homeostasis, but the low proliferative speed of epithelial expansion and reproducibility are the problem. In this review, we hypothesize that to generate cultivated epithelial sheets while maintaining a corneal epithelial stem cells' niche is a better way to proceed as it includes the advantages of both the cell suspension and explant methods. The growth potential, colony-forming efficiency of p63-positive cell clusters, and immunostaining by differentiation/ undifferentiation markers, demonstrated that progenitor cells could be maintained by these gentle enzymatic digestion maintaining cell clusters.     

Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment

Corneal stroma-derived mesenchymal stem cells(CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells(LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.     

Corneal development, limbal stem cell function, and corneal epithelial cell migration in the Pax6(+/-) mouse

PURPOSE: To investigate the etiology of corneal dysfunction in the Pax6(+/-) mouse model of aniridia-related keratopathy. METHODS: Mosaic patterns of X-gal staining were compared in the corneal and limbal epithelia of female Pax6(+/-) and Pax6(+/+) littermates, age 3 to 28 weeks, hemizygous for an X-linked LacZ transgene, and Pax6(+/+), LacZ(-)<-->Pax6(+/+), LacZ(+) and Pax6(+/+), LacZ(-)<-->Pax6(+/-), LacZ(+) chimeras. Histologic examination of chimeric corneas was performed. RESULTS: Disrupted patterns of X-gal staining showed that heterozygosity for Pax6 perturbed clonal patterns of growth and development in the corneal and limbal epithelium. Centripetal migration of Pax6(+/-) corneal epithelial cells was diverted. Normal patterns of centripetal Pax6(+/-) cell migration and epithelial morphology were restored in Pax6(+/+)<-->Pax6(+/-) chimeras. Fewer, larger clones of limbal stem cells were present in Pax6(+/-) eyes, compared with wild-type. In the chimeras, Pax6(+/-) limbal stem cells were cell-autonomously depleted or less efficient than wild-type cells at producing progeny to populate the corneal epithelium. CONCLUSIONS: The correct Pax6 dosage is necessary for normal clonal growth during corneal development, normal limbal stem cell activity, and correct corneal epithelial cell migration. Disruption of normal cell movement in heterozygotes may be the consequence of failure of nonautonomous guidance cues. Degeneration of the corneal surface in aniridia-related keratopathy relates to both a deficiency within the limbal stem cell niche and nonautonomous diversion of corneal epithelial cell migration.     

Long-term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency due to chemical burns

Background: Chemical burns cause depletion of limbal stem cells and eventually lead to corneal opacity and visual loss. We investigated the long‐term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency. Design: Prospective, non‐comparative interventional case series. Participants: Sixteen eyes from 16 patients with severe, unilateral limbal stem cell deficiency caused by chemical burns. Methods: Autologous ex vivo cultured limbal stem cells were grafted onto the recipient eye after superficial keratectomy. Main Outcome Measures: Clinical parameters of limbal stem cell deficiency (stability/transparency of the corneal epithelium, superficial corneal vascularization and pain/photophobia), visual acuity, cytokeratin expression on impression cytology specimens and histology on excised corneal buttons. Results: At 12 months post‐surgery, evaluation of the 16 patients showed that 10 (62.6%) experienced complete restoration of a stable and clear epithelium and 3 (18.7%) had partially successful outcomes (re‐appearance of conjunctiva in some sectors of the cornea and instable corneal surface). Graft failure (no change in corneal surface conditions) was seen in three (18.7%) patients. Penetrating keratoplasty was performed in seven patients, with visual acuity improving up to 0.8 (best result). For two patients, regeneration of the corneal epithelium was confirmed by molecular marker (p63, cytokeratin 3, 12 and 19, mucin 1) analysis. Follow‐up times ranged from 12 to 50 months. Conclusions: Grafts of autologous limbal stem cells cultured onto fibrin glue discs can successfully regenerate the corneal epithelium in patients with limbal stem cell deficiency, allowing to perform successful cornea transplantation and restore vision.