角质细胞生长因子促人角膜上皮细胞生长的研究

目的寻找促进角膜上皮损伤修复的有效方法。方法用３Ｈ胸腺嘧啶核苷（３ＨＴｄＲ）掺入及液体闪烁技术，观察角质细胞生长因子（ＫＧＦ）对体外培养的人角膜上皮细胞ＤＮＡ合成的影响，并计算细胞倍增时间。结果１～１００ｎｇ／ｍｌＫＧＦ有明显促进人角膜上皮细胞ＤＮＡ合成的作用，且呈剂量依赖性（ｒ＝０．９２３３，Ｐ＜０．００１）。１０ｎｇ／ｍｌＫＧＦ明显缩短了细胞倍增时间（３１．５９±４．８８ｈ，与对照组４０．９８±５．２０ｈ比较，Ｐ＜０．０５）。结论外源性ＫＧＦ对体外培养的人角膜上皮细胞有明显的促细胞增生作用。表明ＫＧＦ具有应用于临床，促角膜上皮损伤修复的可能性。     

角质细胞生长因子促进角膜上皮损伤修复的研究

目的寻找促进角膜上皮损伤修复，治疗持续性角膜上皮缺损的有效方法。方法用３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入及液体闪烁技术，观察不同浓度的外源性角质细胞生长因子（ｋｅｒａｔｉｎｏｃｙｔｅｇｒｏｗｔｈｆａｃｔｏｒ，ＫＧＦ）对体外培养的人角膜上皮细胞生长的影响，由此推算出有效滴眼液浓度并应用于兔眼。用计算机图形分析系统计算角膜上皮愈合速率；用光镜和电镜评估愈合的质量。结果０．１～１００ｎｇ／ｍｌＫＧＦ有明显促进体外培养的人角膜上皮细胞生长的作用（增长率为２７．６６％～７６．７３％），且呈剂量依赖性（ｒ＝０．９２３３，Ｐ＜０．００１）。１μｇ／ｍｌＫＧＦ滴兔眼，加速了角膜上皮损伤修复（愈合速率，ＫＧＦ组为１．７７±０．２３ｍｍ２／ｈ，与对照组１．４９±０．２４ｍｍ２／ｈ比较，Ｐ＜０．０５）。结论外源性ＫＧＦ对体外培养的人角膜上皮细胞有明显的促生长作用，其滴眼液有加速兔眼角膜上皮创伤修复的作用。     

青光眼滤过性手术中一次应用5－FU的近期效果

青光眼滤过性手术中一次应用５－ＦＵ的近期效果［英］／ＬａｎｉｇａｎＬ…ＢｒＪＯｐｈｔｈａｌｍｏｌ．－１９９４，７８（１）．－３３～３７青光眼滤过术后结膜下多次注射５－ＦＵ（５－氟尿嘧啶）较烦琐且易引起角膜上皮缺损；而术中一次应用丝裂霉素Ｃ（ＭＭＣ）有...     

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。     

角膜干细胞增殖与分化表达实验研究

目的 了解角膜缘干细胞体外生长的增殖分化特性,检测上皮细胞生长因子对干细胞增殖的促进作用。方法 采用ＤＭＥＭ和Ｆ１２培养基进行兔角膜上皮细胞培养,用克隆形成率（ＣＦＥ）、免疫组化染色和蛋白质免疫印迹等方法检测细胞的增殖力和分化表达状况。结果角膜缘干细胞（ＬＣ）在本培养条件下能够正常传代生长５代,ＣＦＥ为（５．０７±２．３５）％,而角膜中央上皮细胞（ＣＣ）仅第１次传代生长,ＣＦＥ为（１．１２±０．８     

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]     

大鼠角膜缘上皮细胞培养与同体移植的实验研究

目的：观察以明胶为载体培养的角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症的疗效。 方法：大鼠角膜缘上皮细胞在铺有明胶载体的细胞培养板上进行培养5d后,角膜上皮细胞移植术前24h用3H胸腺嘧啶核苷标记培养的角膜缘上皮细胞,培养标记的角膜缘上皮细胞对角膜缘干细胞缺乏的大鼠动物模型行角膜缘上皮细胞同体移植术,对移植术后角膜进行观察、病理学检查及同位素检测。 结果：大鼠角膜缘上皮细胞可以在明胶载体上培养、增殖、分化为密集角膜上皮细胞层;角膜缘上皮细胞移植术后角膜上皮完整、基质细胞浸润减轻、新生血管减少。病理学检查角膜缘及周边部上皮细胞为多层结构,角膜新生血管消失及基质中炎性细胞浸润减轻。角膜缘上皮细胞移植术后4wk受眼角膜仍可测到3H胸腺嘧啶核苷。 结论：角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症可恢复角膜缘干细胞缺乏病变角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘干细胞的屏障功能,为角膜移值提供更好的条件。     

角膜上皮干细胞定位特征的免疫组织化学研究

利用单克隆抗体ＡＥ５与分化型角膜上皮细胞中角蛋白Ｋ３特异性结合,研究缺乏分化标志特征的角膜上皮干细胞定位特点,应用免疫组织化学方法显示Ｋ３阳性表达的区域分布于除角膜缘上皮基底部以外所有角膜上皮细胞中,角膜上皮干细胞存在于角膜缘基底部ＡＥ５抗体反应阴性细胞中,即角膜干细胞位于角膜缘上皮层基底部。     

γ—干扰素对人角膜基质细胞I,Ⅱ型胶原合成的抑制作用

初步探讨γ－ＩＦＮ如何抑制人角膜基质细胞Ｉ，Ⅲ型胶原的合成，用体外培养的２－３代人角膜基质细胞在亚汇合状态下分别加入０．５，５，５０，５００和５０００Ｕ／ｍｌ的γ－ＩＦＮ，共培养４８小时，５００Ｕ／ｍｌ，共培养１２，２４，４８小时，地塞米松作为对照，应用Ｉ，Ⅲ型胶原和纤维连接蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ，ＦＮ）ｃＤＮＡ探针行原位杂交检测ｍＲＮＡ的表达。结果表明，Ｉ，Ⅲ型胶原ｍＲＮＡ的表达与γ－ＩＦ     

角膜上皮干细胞定位特征的免疫组织化学研究

利用单克隆抗体ＡＥ５与分化型角膜上皮细胞中角蛋白Ｋ３特异性结合,研究缺乏分化标志特征的角膜上皮干细胞定位特点,应用免疫组织化学方法显示Ｋ３阳性表达的区域分布于除角膜缘上皮基底部以外的所有角膜上皮细胞中,角膜上皮干细胞存在于角膜缘基底部ＡＥ５抗体反应阴性细胞中,即角膜干细胞位于角膜缘上皮层基底部。     

Comparison of cell-suspension and explant culture of rabbit limbal epithelial cells

Currently, most investigators directly use limbal explants to culture corneal epithelial cells. However, it has not been identified that limbal stem cells do readily migrate from the limbal explants onto culture plate or amniotic membrane carrier. In this study a cell-suspension culture system for rabbit limbal stem cells was developed and compared with the direct explant method in the aspect of stem cells content in the culture system. Rabbit limbal epithelial cells were dissociated from rabbit eyes by dispase and single cell suspension was made for cell-suspension culture. DeltaNp63 expression of cultured rabbit limbal epithelial cells by cell-suspension technique and explant technique was detected. In cell-suspension culture, isolated cell-suspension was evaluated by flow cytometric analysis for vimentin expression and residual limbal tissue after dispase treatment was examined by scanning electron microscopy. In limbal epithelial cells suspension less than 5% cells were vimentin positive. Examination of residual limbal tissue confirmed that all the limbal epithelial cells had been removed. Histological examination revealed that with cell-suspension culture the cultured epithelial cells could differentiate better than with explant technique. In cells cultured with cell-suspension, there were much more cells expressing DeltaNp63 than in explant cultured cells. In cells cultured with explants, most of DeltaNp63 labelling cells mustered around the explants, and peripheral cells on the slides were DeltaNp63 negative. These results suggested that with pure limbal epithelial cells suspension including basal cells, which could directly enter into culture system, cell-suspension culture technique was significantly superior to explant culture technique in terms of stem cells content.     

Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells

Background We have previously developed a limbal epithelial culture system using a cell-suspension method on denuded amniotic membrane (AM). However, other workers reported that intact AM is advantageous for limbal epithelial culture in that it preserves stem cell characteristics. In this study, we cultivated human limbal epithelial cell-suspensions on both intact and denuded AM and compared the morphology and adhesion of the limbal epithelial cells on these two substrates.Methods Human limbal epithelial cells were dissociated from donor eyes using dispase and gentle pipetting and then seeded onto intact and denuded AM as cell suspension. Limbal epithelial cells on AM were co-cultured with a MMC-treated 3T3 fibroblast feeder layer and epithelial differentiation was promoted by air lifting. Cultures were examined by light, scanning and transmission electron microscopy and differences in cellular attachments and intercellular spacing were quantified. Basement membrane complexes were examined by indirect immunofluorescence.Results Limbal cells grown on denuded AM were well stratified and differentiated. Cells were well attached to each other and to the basement membrane. In contrast, limbal cells cultured on intact AM failed to stratify and in places formed a monolayer.The culture on denuded AM had significantly (P<0.001) more desmosomal junctions as well as significantly (P<0.001) more junctional attachments to the carrier than the intact culture. In addition, the intercellular spaces between cells cultivated on denuded AM were significantly (P<0.001) smaller than those between cells grown on the intact substrate. In cultures on both denuded and intact AM, the basement membrane zone displayed a positive staining for collagen VII, integrins alpha-6 and beta-4 and laminin 5.Conclusions We successfully cultivated well-stratified and -differentiated limbal cells on denuded AM, while on the intact AM limbal cells failed to stratify and in places formed only a monolayer of cells. The limbal cells cultivated on denuded AM were well attached to the AM stroma and were morphologically superior to the limbal epithelium cultivated on intact AM. We conclude that for purposes of transplantation of differentiated epithelial sheets, denuded AM is probably the more practical carrier for human limbal epithelial cell cultures when using our cell-suspension culture system.     

胎儿角膜缘干细胞的培养和生物学特性观察

角膜缘干细胞(limbalstemcells,LSC)及利用角膜缘干细胞移植(limbalstemcellstransplantation,LSCT)重建眼表的研究正在国内外广泛展开。而实行LSCT其前提是必须有LSC供体,尽管当前LSC的来源有自体、同种异体和体外培养的自体及同种异体等几种,但均有其局限性。人胎儿细胞的增殖和分化潜能较成人同类细胞大,此外,胎儿的免疫系统没有发育完善,胎儿组织存在免疫宽容现象[1]。贾卉等[2]研究还发现,胎儿角膜中与免疫反应有关的细胞和细胞间黏附分子较成人少,故胎儿器官、组织或细胞移植较少甚至无免疫排斥反应发生。因此,为探寻一种新的…     

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.     

The phenotype of limbal epithelial stem cells

PURPOSE: The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. METHODS: RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes. RESULTS: Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes. CONCLUSIONS: For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.     

Epithelial cell characteristics of cultured human limbal explants

AIM: To determine the immunohistochemical characteristics of putative corneal epithelial stem cells remaining on limbal explants maintained in culture. METHODS: Human limbal explant cultures were generated from 25 residual corneoscleral donor rims following penetrating keratoplasty. Serial sections of these explants were studied using immunohistochemical techniques with a panel of antibodies, on day 0 and 1, 2, and 3 weeks. RESULTS: The number of epithelial cells expressing cytokeratin 19 and vimentin increased with duration in culture, while the number of cells expressing cytokeratin 3 decreased. Connexin 43 expression was lost by 1 week in culture. p63 was expressed by cells that had migrated around the explant and the number of p63 positive cells decreased with longer duration in culture. The explants were initially negative for Ki67, but the epithelial cells were positive at 1 week, and expression of Ki67 was progressively lost with increasing duration in culture. The initial uniform staining of the epithelium for epidermal growth factor receptor and alpha enolase remained unchanged at 3 weeks. CONCLUSIONS: There is an expansion of less differentiated (cytokeratin 3 negative and CK19/vimentin positive) epithelial cells on corneoscleral explants maintained in culture for 3 weeks. The pattern of expression of p63 noted in this study does not support the suggestion that it is a marker of limbal stem cells. The decline in p63 and Ki67 expression among the epithelial cells of the cultured explant button implies that as the epithelial sheet outgrowing from the explant button reaches confluence, the proliferative status of the cells remaining on the explant button declines. These findings are of clinical relevance as explants of limbal tissue are used in limbal stem cell transplantation. There is no information available to date on the fate of epithelial cells on such explants. This study provides some insight into this and suggests that an expansion of the stem cell pool or its progeny may occur in limbal explants.     

Inhibition of vascular endothelial cell morphogenesis in cultures by limbal epithelial cells.

PURPOSE: To study the in vitro angiogenic activity of human conjunctival and limbal epithelial cells and conjunctival, limbal, and corneal fibroblasts in a three-cell-type coculture model. METHODS: Human umbilical vein endothelial cells (EC) were cocultured with epithelial cells, fibroblasts, or epithelial cells and fibroblasts to test their effect on EC morphogenesis. Neutralizing antibodies to some known angiogenic factors were added to the culture to see whether the EC morphogenesis may be blocked by a particular antibody. RESULTS: Conjunctival and limbal epithelial cells exhibited very little or no stimulatory effect on EC tube formation when examined in an EC- epithelial cell coculture system. In contrast, conjunctival, limbal, and corneal fibroblasts all promoted EC morphogenesis when examined under the same culture conditions. Fibroblast-induced EC morphogenesis was inhibited by addition of anti-vascular endothelial growth factor (VEGF) and/or anti-basic fibroblast growth factor (bFGF) antibodies to the culture medium. In the three-cell-type coculture system consisting of ECs, fibroblasts, and epithelial cells, limbal epithelial cells (but not conjunctival epithelial cells) exhibited a strong inhibitory effect on fibroblast-induced EC tube formation. CONCLUSIONS: The proangiogenic activity of ocular surface fibroblasts is probably mediated through a paracrine mechanism by VEGF and bFGF. Limbal epithelial cells, but not conjunctival epithelial cells, inhibit fibroblast-stimulated angiogenesis.     

An evaluation of cultivated corneal limbal epithelial cells,using cell-suspension culture

PURPOSE: A previous report has described an ocular surface reconstruction method involving the use of cultivated corneal epithelium derived from limbal explants. In the current study, a new culture system was developed involving the in vitro propagation on amniotic membrane (AM) of epithelial cells from enzymatically dissociated limbal epithelium. The purpose of this new method is to produce a cultivated epithelial cell layer that contains stem cells and that is superior to explanted cultivated epithelium. The new cell-suspension technique was compared with the existing explant method. METHODS: Limbal epithelial cells were dissociated from donor eyes by dispase and seeded on the denuded AM. Small pieces of limbal epithelium were also cultured on denuded AM as explant cultures. The cultivated epithelium was examined by electron microscopy and immunohistochemistry for cornea-specific keratins (K3 and K12). RESULTS: Both cell-suspension and explant culture methods produced a healthy epithelial cell layer. The cell-suspension culture had significantly (P < 0.001) more desmosomal junctions between the explant-cultured basal cells. In addition, the intercellular spaces between the cell-suspension's basal cells were significantly (P < 0.001) smaller than those between the explant-cultured basal cells. Both types of cultivated epithelium showed positive expression of K3 and K12 keratins. In the cell-suspension culture, expression of K3 and K12 keratins was more prominent in the superficial cells. CONCLUSIONS: Corneal epithelial cells were successfully regenerated in vitro by a cell-suspension culture system. The suspension-cultured epithelium must include some stem cells and morphologically is significantly superior to explant-cultured epithelium. Thus, this new technique is potentially more suitable for cultivated corneal limbal epithelial transplantation.     

Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.