角膜缘微环境细胞的研究进展

角膜缘微环境细胞（limbal niche cell,LNC）是近年来发现的一种来自于角膜缘干细胞微环境且具有血管内皮祖细胞和间充质干细胞特性的多能干细胞。LNC具有分化为血管内皮细胞、角膜上皮细胞、角膜基质细胞等多种细胞的潜能。体外实验发现,LNC在三维立体培养环境中可以维持角膜缘干细胞的特性;动物实验发现,LNC可以预防和治疗碱烧伤所致的角膜缘干细胞缺乏。LNC还可以促进角膜基质的无瘢痕修复,减少修复过程中的新生血管化,提示其可以重建角膜基质。因此,在角膜缘干细胞缺乏、角膜基质损伤等疾病的治疗中,LNC可能是一种理想的治疗细胞。     

角膜基质诱导胚胎干细胞定向分化的初步实验研究

目的:探索角膜基质接触诱导胚胎干细胞(embryonic stem cells,ES.细胞)定向分化的可能性。方法:分别在去上皮的新西兰白兔表层角膜缘基质上、晶状体上皮细胞饲养层上培养ES-D_3细胞,裸鼠皮下移植使其形成复层细胞,一段时间后,行扫描电镜和光镜观察。结果:I.ES细胞在新鲜表层角膜缘基质上增殖缓慢,2～3周形成较大细胞集落,光镜下细胞形态单一,体积较正常ES细胞大,电镜下细胞核已演变为细长形,移植到裸鼠皮下2周形成上皮样细胞复层,电镜下可见微绒毛,而角膜基质非上皮面的ES细胞仍保持其小细胞状态不变。2.晶状体上皮细胞与ES细胞共培养只能延缓其分化时间,不能诱导其定向分化。结论:表层角膜缘基质具有诱导ES细胞定向分化的潜能,体外培养条件下可能不发生晶状体诱导的第三级胚胎诱导。眼科学报1999;15:195-198。     

培养角膜缘上皮细胞深低温保存后异体移植实验研究

目的深低温保存培养的角膜缘上皮细胞,复苏后进行异体移植实验,为临床应用提供实验依据。方法将浅层角膜基质载体上培养的角膜缘上皮细胞深低温保存,同时制作兔角膜缘干细胞损伤模型,分别用新鲜培养的角膜缘上皮细胞和冷冻复苏后培养的细胞及角膜基质进行异体移植实验。结果角膜缘上皮细胞能成功地培养于载体上,将培养细胞保存于-196℃的液氮罐中二月,可见深低温保存前后细胞的形态结构及生物学特性无显著差异。异体移植实验显示：新鲜培养的细胞及深低温保存后的细胞异体移植后,模型兔角膜表面逐渐正常上皮化,而角膜基质组异体移植后,角膜表面仍结膜化并可见大量新生血管生长,各项检测结果与培养细胞组相比差异显著（P〈0．05）。结论以浅层角膜基质为载体培养的角膜缘上皮细胞深低温保存后仍具有上皮细胞特性并具有高增殖能力,可用以进行异体移植治疗角膜缘干细胞缺陷的角膜病。     

Surgical approaches for limbal stem cell deficiency

BACKGROUND: Stem cells are the ultimate source of the rapidly self-renewing corneal epithelium and are located in the basal layer of the limbal epithelium. A variety of diseases can compromise the stem cell pool, causing an entity called limbal stem cell deficiency. Several therapeutic strategies should be taken into account. METHODS AND RESULTS: Ocular surface defence should be restored prior to any kind of stem cell transplantation to assure a sufficient and stable tearfilm. Surgical strategies comprise superficial keratectomy with or without amniotic membrane transplantation, conjunctival-limbal autograft and keratolimbal allograft transplantation. A damaged limbal and corneal stroma can be reconstructed by amniotic membrane as a biological substrate. First encouraging results have been reported using tissue bio-engineered limbal epithelium on amniotic membrane for corneal surface reconstruction. CONCLUSION: The correct diagnosis and a restored ocular surface defence are a prerequisite for subsequent transplantation of limbal stem cells. Optimal patient selection for the respective surgical approach and systemic immunosuppression protocols to improve allograft survival need to be further investigated.     

以干燥脱水法保存的异种角膜基质为载体构建人工生物角膜上皮组织

目的:观察以干燥脱水法保存的鸵鸟角膜基质为载体构建人工生物角膜上皮组织的生物学特性。方法:采用组织块培养法获得新西兰大白兔角膜缘干细胞,经胰蛋白酶消化法获得细胞,种植于干燥脱水法保存的鸵鸟角膜板层基质上,采用气液界面培养法进行培养,通过倒置显微镜、透射电子显微镜、荧光显微镜观察其形态学、生长特点,超微结构及免疫学特征。结果:在干燥脱水法保存的鸵鸟角膜基质上种植兔角膜缘干细胞,接种72h后,细胞形成单层,移置气液交界面后继续培养7～10d,逐渐形成复层。经光镜、透射电镜、及免疫学检测显示其具有角膜上皮组织的生物学特性。结论:兔角膜缘干细胞能够在干燥脱水法保存的鸵鸟角膜基质载体上生长,并可形成复层,基本具有正常角膜上皮细胞的形态、超微结构和生物学特性。     

Combined procedures in glaucoma surgery

The corneal limbus harbors corneal epithelial stem cells and contributes to the unique microenvironment of the stem cell niche. Corneal conditions, such as infections, tumors, immunological disorders, trauma, and chemical burns, often lead to the deficiency of the corneal stem cells, and subsequent vision loss. One key feature of limbal stem cell deficiency is corneal neovascularization. There is a delicate balance between pro-angiogenic and anti-angiogenic factors that, in a normal cornea, maintain an avascular state. A pro-angiogenic shift in this balance can occur due to various mechanisms, such as inflammation, gene mutations, physical breach in the limbal barrier, and decreased production of anti-angiogenic molecules. Currently available treatment options for limbal stem cell deficiency include allogeneic and autologous limbal transplants, and more recently, transplantation of alternative sources of epithelium, such as cultivated corneal and oral mucosal stem cells. Further studies are needed to investigate the combination of limbal and stem cell transplantation and concurrent anti-angiogenic therapy.     

角膜缘组织定位培养和冷冻后培养的实验研究

目的验证角膜缘干细胞的组织学定位,探讨低温冷冻保存对其增殖活性的影响。方法取新鲜角膜缘上皮组织和相应部位浅层巩膜组织各10块进行体外细胞培养,对比观察细胞生长情况。取冷冻保存的角膜缘上皮组织14例,观察体外培养后细胞生长情况。通过免疫组化方法检测冷冻保存的角膜缘上皮细胞的增殖活性和培养后单层细胞K3角蛋白的表达。结果10例新鲜角膜缘上皮组织培养后,7例有上皮细胞生长,1周形成细胞单层;10例浅层巩膜组织培养后未见细胞生长。14例冷冻角膜缘上皮组织培养后,4例有上皮细胞生长,9d形成细胞单层。5例冷冻角巩膜环组织冰冻切片中,3例可见角膜缘上皮基底细胞PCNA表达阳性。培养细胞对K3角蛋白特异性的AE-5单克隆抗体免疫反应阳性。结论角膜缘干细胞定位于角膜缘上皮基底部,低温冷冻保存的角膜缘干细胞组织可以保持增殖活性,体外培养后生长分化成为角膜上皮。     

增龄对角膜基质载体培养的角膜缘干细胞生物学特性的影响

目的 观察增龄对新西兰兔角膜缘干细胞在同种异体角膜基质上生长、增殖的影响.方法 用酶消化法将不同年龄新西兰兔角膜缘组织制成细胞悬液培养,以相同的细胞密度种植于不同年龄的新西兰兔的角膜基质上,观察角膜缘干细胞在角膜幕质上的生长情况.原位杂交检测ABCG2、ANp63的表达,IPP5.1软件分析图像,流式细胞术测细胞周期,运用SPSS13.0软件对析因设计资料进行方差分析.结果 角膜缘干细胞供体年龄各水平差异有统汁学意义,相同时间内角膜基质培养的青年兔角膜缘干细胞在角膜基质单位面积卜的牛长总而积均较中年和老年高,增殖期细胞所占比例大;而角膜基质供体年龄各水平差异与角膜缘干细胞和角膜基质的交互作用均无统计学意义.结论 在同种异体角膜基质载体上培养的角膜缘干细胞的增殖能力随着年龄的增加而降低.     

Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche

Identification, maintenance, and expansion of stem cells for subsequent transplantation has become a new strategy for treating many diseases in most medical subspecialties. The stem cells of the corneal epithelium are located in the limbal basal layer and are the ultimate source for constant corneal epithelial renewal. Like those in other tissues, limbal stem cells are supported by a unique stromal microenvironment called the stem cell niche, which consists of certain extracellular matrix components, cell membrane-associated molecules, and cytokine dialogues. Destructive loss of limbal stem cells or dysfunction of their stromal environment renders many corneas with a clinical entity called limbal stem cell deficiency, which is characterized by variable extents of conjunctival ingrowth depending on the severity of limbal damage. A new strategy of treating limbal stem cell deficiency is to transplant a bio-engineered graft by expanding limbal epithelial stem cells ex vivo on amniotic membrane. This review summarizes the published literature data collectively explaining how amniotic membrane is an ideal biological substrate that can help maintain and support the expansion of limbal epithelial stem cells.     

角膜缘干细胞的研究

角膜缘干细胞是位于角膜缘基底上皮层底的一类特殊类型的上皮细胞,随着细胞培养技术的发展,角膜缘干细胞体外培养后移植用于治疗由于角膜缘干细胞缺乏或者功能不全引起的眼表疾病成为研究的热点。本文就其解剖学定位、生物学特性、组织工程化角膜的基础性研究及其临床应用做一综述。     

Stem cells, wound healing, growth factors, and angiogenesis in the cornea

The integrity of the corneal epithelium is, among other factors, dependent on the function of corneal stem cells which serve as proliferative reserve. The concept that corneal stem cells are located in the basal limbal epithelium has been further strengthened by experimental evidence. Furthermore, the usefulness of surgical treatment for bilateral stem cell malfunction by means of limbal allograft transplantation has been confirmed. The field of growth factor interactions has further been expanded by the meaning such factors have for the paracrine cross-signaling between the various corneal cell populations, especially epithelium and stroma. This review focuses on the progress that has been made in the field of corneal cell biology in the context of stem cells, wound healing, and corneal angiogenesis.     

Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle

Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells.     

The corneal epithelial stem cell niche

In recent years, it has become generally accepted that the corneal epithelial stem cells are localized in the basal cell layer of the limbal epithelium. However, a number of questions remain regarding the number, markers, generation, and maintenance of the corneal epithelial stem cells. One of the key questions concerns what makes up the microenvironment or niche that is responsible for allowing the stem cells to remain and function throughout the life of the tissue. This review will consider the unique aspects of the limbus and compare these to what is known about other stem cell niches.     

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)     

Biologische Grundlagen der Limbusstammzellinsuffizienz

Limbal stem cell deficiency results from the loss of tissue regenerating stem and progenitor cells. Corneal epithelial regeneration is maintained by stem and progenitor cells which reside in the schlerocorneal limbus. They possess stem cell characteristics and can be stimulated to proliferate by external signals. The limbus is the stem cell niche for corneal epithelial stem cells and forms a unique microenvironment in which stem cell characteristics are conserved. Regulation of limbal epithelial stem cells is produced by a network of signals within the niche which governs cell fate decisions with regards to proliferation, differentiation or maintenance of a quiescent status.     

Oberfl?chenrekonstruktion bei Limbusstammzellinsuffizienz

Various ocular surface diseases are caused by loss of corneal epithelial stem cells or dysfunction of the limbal stem cell niche. Besides conventional transplantation of autologous or allogenic limbal tissue, recent advances in tissue engineering have led to the development of new culture and expansion techniques of human limbal stem and progenitor cells (LSPC) as a new strategy to successfully treat limbal stem cell deficiency (LSCD). From a small autologous limbal biopsy with a limited amount of LSPC an epithelium ready for transplantation is achieved. Autologous grafting of cultured limbal epithelium led in most of the treated cases to a successful reconstruction of the corneal surface. Alternative methods which have recently been introduced to treat LSCD use other stem cell sources including the transplantation of oral mucosal epithelium. In this article the challenges and controversies associated with these stem cell culture techniques for ocular surface reconstruction are reviewed.     

角膜缘niche细胞与基质细胞维持角膜缘干细胞功能的对比研究

目的　比较角膜缘niche细胞（limbal niche cells，LNCs）与角膜缘基质细胞（limbal stromal cells，LSCs）在维持角膜缘干细胞功能上的不同特性。方法　将LNCs和LSCs分别从6个角膜缘组织分离，并在相同的条件下培养、传代。LNCs与LSCs经丝裂霉素C（mitomycin C,MMC）处理后分为LNCs组与LSCs组作为饲养细胞分别与角膜缘干细胞共培养，比较两组角膜缘干细胞克隆形成率（colony-forming efficiency,CFE）、上皮细胞复层化以及细胞标志物和部分基因的表达。结果　LNCs组角膜缘干细胞CFE（6.57±1.54）%高于LSCs组（1.43±0.47）%。 LNCs组细胞复层上皮数（4~5层）多于LSCs组（2~3层）。角膜缘干细胞克隆与免疫荧光染色及mRNA半定量分析结果显示，LNCs组比LSCs组表达了更多干细胞标志物ΔNp63，能更有效地维持角膜缘干细胞的细胞特性。逆转录PCR分析结果显示，LNCs组与LSCs组都分泌了一些维持角膜缘干细胞生长的生长因子，但LNCs组比LSCs组高表达上皮型钙黏蛋白（E-cadherin），低表达营养神经素3（NT3），能更好地支持角膜上皮增殖。结论　LNCs比LSCs能更好地支持角膜缘干细胞的生长及维持其干细胞特性。     

Mesenchymal stem cells derived from human limbal niche cells

Purpose. We investigated whether human limbal niche cells generate mesenchymal stem cells. Methods. Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells. Results. Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α. Conclusions. In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.     

羊膜移植联合鸡角膜缘上皮移植治疗兔全角膜表层损伤的组织学改变

目的：研究羊膜移植术和联合鸡角膜缘上皮移植（联合移植）术治疗兔全角膜表层损伤的组织学改变。方法：兔39只分为3组，每组13只。对照组，将角膜及角膜缘外3mm的表层组织去除；羊膜移植组，在去除表层组织的创面上移植羊膜；联合移植组，在羊膜移植片表面角膜缘区移植鸡角膜缘上皮。手术当天各组处死1只兔，7、28、90、105天各组处死3只兔，摘除眼球分别进行光镜、免疫组化，透射电镜及扫描电镜检查。结果：对照组：角膜紊乱，表面不平，上皮表面微绒毛稀少，基质纤维血管化。羊膜移植组及联合移植组：羊膜结构逐渐被基质胶元纤维取代和改建，基质纤维平行排列，角膜表面光滑，上皮表面微绒毛丰富，细胞之间可见桥粒连接。结论：羊膜移植术和联合鸡角膜缘移植术治疗兔角膜广泛损伤，能抑制新生血管和纤维组织向角膜生长，较快促进眼表重建。