间充质干细胞在角膜重建中的应用研究进展

角膜重建指的是各种外伤、炎症、变性性疾病引起的角膜上皮损伤、新生血管形成、角膜瘢痕等损伤之后,角膜结构完整性及角膜透明性的恢复。目前角膜重建的基础研究中,间充质干细胞逐渐成为研究的热点。本文对间充质干细胞在角膜重建中作用及进展予以综述,将为角膜重建的深入研究提供一定的参考资料。     

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。     

干细胞与角膜眼表重建研究进展

角膜是眼表的重要防护屏障和光学系统的重要组成部分,其完整性和透明性是实现有效视功能的重要前提。各种原因造成的眼表疾病未经及时有效治疗都有可能发展到终末阶段———角膜缘干细胞功能障碍,包括角膜缘干细胞数目的减少和微环境的病理改变。自体角膜移植无疑是治疗角膜缘干细胞功能障碍最有效的方法。但是供体角膜来源十分有限,且切除健眼组织行角膜移植可能会对健眼造成长期损害。随着干细胞研究的不断深入和组织工程技术的兴起,组织工程角膜应运而生,并且迅速发展。其基本原理为选用生物性能良好的支架材料,体外模拟角膜缘干细胞微环境,诱导各类种子干细胞分化为角膜类上皮,然后再行角膜眼表重建。常用种子细胞包括角膜缘干细胞、胚胎干细胞、诱导多能干细胞、骨髓间充质干细胞、皮肤干细胞、口腔黏膜干细胞。本文就上述干细胞在角膜眼表重建中的应用作一综述。     

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。     

干细胞移植与角膜功能重建的研究进展及其存在的问题

近年来,干细胞移植,包括自体或异体角膜缘干细胞、口腔黏膜上皮以及体外培养的干细胞移植进行角膜功能重建的基础研究取得了一些进展,在临床应用中也得到了较好的治疗效果,但对眼表严重损伤的治疗效果、远期治疗评价和移植细胞的体内转归方面仍无定性的结论。对体外培养的干细胞移植重建角膜功能的基础研究和临床应用方面的研究进展进行介绍和比较,并对存在的问题进行讨论。     

角膜缘干细胞移植治疗角膜缘干细胞功能缺陷的研究进展

健康的角膜缘干细胞是维持角膜上皮透明与完整的前提.各种原因引起的角膜缘干细胞和/或角膜缘干细胞龛受损均可导致角膜缘干细胞功能缺陷(limbal stem cell deficiency,LSCD).角膜缘干细胞移植是恢复干细胞功能、治疗LSCD的主要方法之一.角膜缘干细胞移植主要分为三大类:直接移植角膜缘组织、体外培养...     

角膜缘干细胞的研究进展

健康的角膜上皮是维系完整的眼表以及良好视功能的前提条件,角膜上皮的稳定是由一群位于角膜缘基底部的干细胞实现的。一、角膜缘干细胞的概念Davanger和Evensen[1]于1971年首次观察到角膜缘色素样细胞作水平向心运动,推测角膜上皮细胞更替源于角膜缘,提出了角膜缘干细胞(limbalstem     

培养角膜缘干细胞移植重建眼表的研究进展

角膜缘干细胞缺乏所致眼表疾病已成为角膜病致盲的重要原因之一。随着细胞培养技术的发展,角膜缘干细胞的重要作用已经得到了极大的肯定。角膜缘干细胞体外培养后移植是治疗眼表疾病的重要途径。本文就其组织学定位、培养载体和基础研究做一综述,以便对角膜缘干细胞有更深入的研究。     

人牙周膜干细胞、脐带间充质干细胞对体外培养角膜缘干细胞的影响

目的　比较人牙周膜干细胞（ｈｕｍａｎｐｅｒｉｏｄｏｎｔａｌｌｉｇａｍｅｎｔｓｔｅｍｃｅｌｌｓ,ＨＰＤＬＳＣｓ）、人脐带间充质干细胞（ｈｕｍａｎｕｍｂｉｌｉｃａｌｃｏｒｄｍｅｓｅｎｃｈｙｍａｌｓｔｅｍｃｅｌｌｓ,ＨＵＣＭＳＣｓ）作为饲养层培养角膜缘干细胞（ｌｉｍｂａｌｓｔｅｍｃｅｌｌｓ,ＬＳＣｓ）的优越性,从而筛选出扩增ＬＳＣｓ的最佳饲养层。方法　体外分离培养ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ,诱导细胞成脂、成骨分化,并检测细胞表面标志物的表达;将兔ＬＳＣｓ与ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ、ＮＩＨ-３Ｔ３共培养和无饲养层单独培养,比较各组克隆形成率,免疫荧光比较各组ＬＳＣｓ克隆团ＡＢＣＢ５、ＩＰＯ１３、ＣＫ３／１２的表达情况。结果　体外培养ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ均可向脂肪、成骨细胞分化,两种细胞均高表达间充质来源的表面标志ＣＤ９０,不表达ＣＤ４５;三组饲养层与兔ＬＳＣｓ共培养均可形成小克隆团, ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组、ＮＩＨ-３Ｔ３组、无饲养层组克隆形成率分别为（４．９０±０．９６）％、（４．１０±０．５６）％、（４．６７±０．７６）％、（０．８３±０．３５）％,四组间总体比较差异有统计学意义（Ｆ＝２２．０４７,Ｐ＝０．００）;ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组与ＮＩＨ-３Ｔ３组的克隆形成率差异均无统计学意义（均为Ｐ＞０．０５）;ＨＰＤＬＳＣｓ组、ＨＵＣＭＳＣｓ组、ＮＩＨ-３Ｔ３组与无饲养层组间差异均有统计学意义（均为Ｐ＜０．０１）。免疫荧光化学检测三种饲养层ＬＳＣｓ标志物ＡＢＣＢ５、ＩＰＯ１３呈高表达,ＣＫ３／１２呈低表达。结论　ＨＰＤＬＳＣｓ、ＨＵＣＭＳＣｓ均可作为替代饲养层用以培养ＬＳＣｓ。     

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。     

Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells

AIM: To explore the possibility of human umbilical cord mesenchymal stem cells (hUCMSCs), human umbilical vein endothelial cells (hUVECs), human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs) serving as feeder cells in co-culture systems for the cultivation of limbal stem cells. METHODS: Different feeder layers were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 and were treated with mitomycin C. Rabbits limbal stem cells (LSCs) were co-cultured on hUCMSCs, hUVECs, hDPSCs, hPDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency (CFE) assay and immunofluorescence (IPO13,CK3/12). RESULTS: The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But hUCMSCs, hDPSCs and hPDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to hUVECs and feeder-cell-free culture. CONCLUSION: hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.     

人角膜基质细胞诱导人骨髓间充质干细胞分化为角膜上皮细胞的初步研究

目的 探讨人骨髓间充质干细胞（MSC）体外分化为角膜上皮细胞的可行性.方法 实验研究.分别取第3代人MSC和自行培养的第3代人角膜基质细胞共同培养1周,实验组在Transwell共培养体系中培养,对照组不放置Transwell小室培养.1周后,观察实验组和对照组中人MSC光镜特征、间接免疫细胞化学染色和电镜结构,对被诱导分化的细胞进行综合鉴别.结果 第3代人MSC和人角膜基质细胞在体外培养条件下均能够较快贴壁生长.两种细胞共同培养1周后,可见部分细胞形态上呈上皮细胞特征,单克隆抗体AE1染色呈阳性,电镜下可见微绒毛、桥粒和张力丝等典型上皮细胞结构特征.结论 体外培养的人MSC在人角膜基质细胞的诱导下可能会分化为人角膜上皮样细胞.     

角膜上皮干细胞的体外生长特征

目的　了解角膜上皮干细胞的生长特性。方法　用体外细胞培养的方法 ,从生长曲线、细胞倍增时间、细胞 DNA合成的活跃程度等几个方面 ,观察人角膜缘部、周边部及中央部角膜上皮细胞的生长特征。结果　角膜缘部细胞群体倍增时间最短 (4 9.79h±1.2 6 h) ,周边部次之 (6 4.89h± 2 .18h) ,中央部最长 (78.86 h± 1.38h) ,3组两两比较 P<0 .0 1;角膜缘部角膜上皮细胞 DNA合成最活跃 (CPM值 92 7.75± 47.94) ,周边部次之(CPM值 711.75± 2 9.47) ,中央部最弱 (CPM值 5 14.0 0± 72 .82 ) ,3组两两比较 P <0 .0 1。结论　结果进一部证实了角膜上皮干细胞存在于角膜缘的观点 ;也反映了角膜缘在角膜上皮创伤愈合 ,角膜上皮完整与稳定性维持过程中起着重要的作用     

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。     

羊膜移植联合角膜缘干细胞移植治疗严重眼烧伤

目的观察羊膜移植联合上下角膜缘干细胞移植治疗严重眼烧伤的效果。方法采用新鲜羊膜移植联合上下角膜缘干细胞移植治疗严重眼烧伤患者15例（17眼）。术后随访平均2月，观察其效果。结果视力大于0．05者12眼占70．59％，10眼角膜无明显新生血管，2眼角膜中央留有薄翳，12眼角膜稍有浑浊。17眼全部瘢痕愈合，眼表稳定。结论烧伤早期行羊膜移植联合上下角膜缘干细胞移植可明显减少睑球粘连并可使早期眼表稳定。     

Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results

Ex vivo cultured limbal epithelial stem cells have been used successfully to treat corneal limbal stem cell deficiency. We identified 17 reports of the application of this novel cell-based therapy in humans. In addition we identified four reports of the use of culture oral mucosal epithelial cells to treat limbal stem cell deficiency. We examined these reports to discern the success rate, complication rate, visual outcome, whether there is an optimal technique and which patients are the most likely to benefit. We also discuss the different culture methods employed and the regulations governing cell banks that are providing this service. We found that the techniques used to cultivate and transplant cells varied, but that no individual method was clearly superior. The reported success rate is similar across all studies for both allografts and autografts. The clinical indications for this treatment are not clearly defined as indicated by the variety of disorders treated. Follow-up is limited and the long-term success rate is yet to be established. Nonetheless, we conclude that there is sufficient evidence to support the continued use and refinement of this procedure as a treatment for corneal stem cell deficiency.     

Identification and characterization of limbal stem cells

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SC) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SC biology remains the ability to identify stem cells in situ and in vitro. Until recently, the identification of limbal stem cells mainly has been based on general properties of stem cells, e.g. lack of differentiation, prolonged label-retaining, indefinite capacity of proliferation exemplified by the clonogenic assay as well as their special role in corneal wound healing. During the last years, a number of molecular markers for the limbal SC compartment has been proposed, however, their role in distinguishing limbal SC from their early progeny is still under debate. Data reported from the literature combined with our own recent observations suggest, that the basal epithelial cells of the human limbus contain ABCG2, K19, vimentin, KGF-R, metallothionein, and integrin alpha9, but do not stain for K3/K12, Cx43, involucrin, P-cadherin, integrins alpha2, alpha6, and beta4, and nestin, when compared to the basal cells of the corneal epithelium. A relatively higher expression level in basal limbal cells was observed for p63, alpha-enolase, K5/14, and HGF-R, whereas there were no significant differences in staining intensity for beta-catenin, integrins alphav, beta1, beta2, and beta5, CD71, EGF-R, TGF-beta-RI, TGF-beta-RII, and TrkA between limbal and corneal basal epithelial cells. Therefore, a combination of differentiation-associated markers (e.g. K3/K12, Cx43, or involucrin) and putative SC-associated markers (e.g. ABCG2, K19, vimentin, or integrin alpha9) may provide a suitable tool for identification of human limbal SC. While most putative SC markers label the majority of limbal basal cells and, therefore, may not distinguish SC from progenitor cells, only ABCG2 was strictly confined to small clusters of basal cells in the limbal epithelium. At present, ABCG2 therefore appears to be the most useful cell surface marker for the identification and isolation of corneal epithelial SC. Moreover, the characteristics of the specific microenvironment of corneal SC, as provided by growth factor activity and basement membrane heterogeneity in the limbal area, could serve as additional tools for their selective enrichment and in vitro expansion for the purpose of ocular surface reconstruction.     

带角膜缘干细胞的自体结膜瓣移植术治疗复发性翼状胬肉

目的:观察带角膜缘干细胞的自体结膜瓣移植术治疗复发性翼状胬肉的临床有效性和实用性。方法:复发性翼状胬肉患者53例57眼。复发性翼状胬肉切除全部病灶组织后移植带角膜缘干细胞的自体结膜瓣,移植结膜瓣取自术眼上方结膜。结果:术眼57眼术后随访1~12mo,3例出现复发,复发率5%。1例发生角巩膜自溶,并手术治疗。余53眼经1~12mo的随访角膜创面愈合良好,植片全部存活,无明显手术痕迹,治愈率93%。结论:采用自体角膜缘干细胞移植术治疗复发性翼状胬肉,手术取材方便、创面修复快,术后复发率低,满足部分患者美观需求,改善了术后效果,是一种理想的术式,值得基层临床推广应用。     

骨髓间充质干细胞移植重建碱烧伤眼表的实验研究

目的 探索人骨髓间充质干细胞(MSCs)的诱导分化条件,观察负载人MSCs的羊膜移植重建碱烧伤大鼠眼表的疗效.方法 传三代人MSCs分组培养,不同条件刺激后种于羊膜表面.碱烧伤后1个月大鼠随机分A、B、C和D组:A组(A1、A2、A3、A4)移植不同条件诱导的人MSCs,B组移植人角膜缘干细胞(LSCs),C组移植羊膜,D组未治疗.术后观察眼表并评分,2周和1个月时行印迹细胞学(IC)检查,1个月时切片行苏木精-伊红染色,免疫组织化学和免疫荧光检测溴脱氧尿嘧啶(BrdU)、ABCG2、K3/K12、Cx43和血管内皮生长因子(VEGF).结果 术后A、B组较C、D组角膜新生血管和炎性细胞减少、透明度增加(P＜0.05).IC检查示A、B组未见杯状细胞,C、D组可见杯状细胞.A、B组均形成3层以上上皮细胞,C组3层左右,D组为1～2层.免疫组织化学和免疫荧光检查示:A、B组角膜表面可检测到BrdU、ABCG2阳性细胞;A组Cx43均为阴性,A4组中有2个标本K3/K12为弱阳性,其余A组标本均为阴性,B组K3/K12、Cx43均为阳性;A、B组VEGF均较低,C组较强,D组最强.结论 人MSCs与人LSCs相同,可成功重建碱烧伤大鼠眼表.其眼表重建功能可能与抗炎和抗血管化作用有关.