翼状胬肉上皮细胞异常表达角蛋白

目的 了解角蛋白在翼状胬肉上皮细胞的表达。方法 应用免疫荧光组织化学染色法检测８例翼状胬肉上皮细胞角蛋白家族的表达,并与４例正常人对比。结果 在正常结膜上皮,Ｋ８,Ｋ１０,Ｋ１６染色极弱或阴性,Ｋ１４及ＡＥ３仅在基底层染色阳性;在翼状胬肉上皮,Ｋ８,Ｋ１４,Ｋ１６及ＡＥ３为全层表达,Ｋ１０在上皮的部分表层细胞中表达。结论 翼状胬肉上皮细胞异常表达角蛋白家族提示其上皮细胞处于增生活跃状态并出现角化现象。     

In vivo survival and stratification of cultured limbal epithelium

A 6-year-old Bangladeshi girl presented with total limbal stem cell deficiency in the left eye, secondary to a 6-month-old chemical injury. The patient had also previously undergone two limbal transplantation surgeries. At the authors' centre the child underwent autologous cultured limbal epithelium transplantation, on human amniotic membrane, without the use of air-lift technique. Symptomatic relief, re-epithelialization of the ocular surface, regression of corneal pannus and slight improvement in vision were all noted. The corneal button obtained at the time of keratoplasty (performed 4 months later) revealed stratified epithelium with basement membrane. Thirty-seven months post keratoplasty, the best-corrected visual acuity was 6/15 with clear graft and stable ocular surface. Herein, a case of limbal stem cell deficiency successfully managed by monolayer of cultured limbal epithelium is presented.     

不同移植手术治疗角膜缘干细胞缺损的实验研究

目的探讨以人羊膜为载体培养的角膜缘干细胞，自体及异体移植治疗全角膜缘干细胞缺损。方法制作兔眼角膜缘干细胞完全缺损3个月的模型。实验动物随机分为自体移植组和异体移植组，前者取对侧眼角膜缘组织，后者取异体兔眼角膜缘组织，均以去除上皮细胞的羊膜基底膜为载体，培养12d后行角膜缘干细胞羊膜移植术。术后观察3个月，以角膜上皮染色、角膜浑浊和新生血管3项指标进行临床疗效评定，通过病理检查评估术后角膜上皮修复情况，印迹细胞学检查移植前后角膜上皮的细胞表型。结果体外培养的兔角膜缘干细胞可在羊膜上粘附生长并增生，体外培养12d可形成复层。自体移植组和部分异体移植组术后角膜上皮逐渐愈合，透明度提高，基质细胞浸润减轻，新生血管减退或消失。印迹细胞学检查显示：移植前角膜上皮细胞PAS阳性，而移植后转为阴性；组织病理学显示：移植前角膜上皮大部分缺损，移植后呈现角膜上皮结构。部分异体移植组术后出现了免疫排斥反应。结论兔自体角膜缘干细胞羊膜移植术可重建眼表；免疫排斥反应仍是异体角膜缘干细胞羊膜移植术失败的主要原因。     

Preoperative evaluation of cultured human corneal limbal epithelium on amniotic membrane by confocal microscopy

PURPOSE: Although sheet transplantation with cultured corneal limbal epithelium has been widely performed as a strategy for ocular surface reconstruction, there has been no optimal method for evaluating the morphology of these sheets prior to transplantation. We propose the use of in vivo confocal microscopy as a novel method for the evaluation of limbal corneal epithelium cultured on amniotic membrane. METHODS: Human limbal epithelial sheets were grown on amniotic membranes by following a standard protocol and were stained with hematoxylin and eosin. Morphology was studied using in vivo confocal microscopy for cultured corneal epithelium on amniotic membrane, human intact amniotic membranes, and epithelium-denuded human amniotic membranes. RESULTS: Histologic examination showed a stratified corneal epithelium sheet by the fourth week of culture. The surface and basal layers of the cultured limbal epithelium and amniotic membrane were clearly distinguished by in vivo confocal microscopy. A monolayer of amniotic epithelial cells was observed on the intact amniotic membrane, but not on the epithelium-denuded human amniotic membrane. CONCLUSIONS: Our findings support the use of in vivo confocal microscopy as a valid technique for the preoperative evaluation of cultured corneal limbal epithelial cell sheets on amniotic membrane.     

Ex-vivo potential of cadaveric and fresh limbal tissues to regenerate cultured epithelium

PURPOSE: To evaluate and compare the ex-vivo growth potential and formation of cultured corneal epithelium from residual corneo-limbal rings obtained from the operating room after penetrating keratoplasty, and fresh limbal tissues from patients undergoing routine cataract surgery. METHODS: With the approval of the Institutional Review Board and informed consent from patients, 1-2 mm of limbal tissues from 15 patients and 31 tissues from the cadaveric limbal ring preserved in MK medium (16 tissues) and Optisol (15 tissues) were used for the study. Donor data included age, time lapse between death and collection, collection and preservation and preservation and culture. Tiny bits of the limbal tissue were explanted on the de-epithelialised human amniotic membrane prepared following standard guidelines, and cultured using Human Corneal Epithelial cell medium. Radial growth from the explant was observed and measured by phase contrast microscopy over 2-4 weeks. After adequate confluent growth, whole mount preparation of the membrane was made and stained with haematoxylin and eosin. Part of the membrane was fixed in formalin and processed for routine histologic examination. The sections were stained with haematoxylin and eosin. RESULTS: Forty-six tissues were evaluated from 42 eyes (15 from patients, 31 from cadaveric eyes) with a mean age of 55.3 years +/- 21.23 years (range 18 years - 110 years). The growth pattern observed was similar in all the positive cases with clusters of cells budding from the explant over 24-72 hours, and subsequent formation of a monolayer over the next 2-3 weeks. The stained whole mount preparation showed a radial growth of cells around explants with diameter ranging from 5 to 16mm. Histologic evaluation of the membrane confirmed the growth of 2-3 cell-layered epithelium over the amniotic membrane. Cultivated epithelium around explant cell cultures was observed in 100% (15/15) of limbal tissue obtained from patients, as against 56% (9/16) of MK medium preserved tissues and 46.7% (7/15) of Optisol preserved tissues. This was statistically significant (P=0.0131) There was no significant statistical difference in the growth properties, i.e, the mean percentage of fragments showing growth (P=0.229) or the mean diameter of growth (P=0.479) in the cultures obtained from fresh and preserved tissues. The time lapse at various stages between death and utilisation and donor age had no significant influence on the growth potential of the limbal tissues. CONCLUSION: The potential for generating cultured corneal epithelium from fresh limbal tissues obtained from living subjects is higher than that observed with preserved tissues. It would also be worthwhile to address the factors that could further enhance the proliferative potential of the cadaveric tissues obtained from eye banks.     

Disease expression in cultured pigment epithelium. Feline mucopolysaccharidosis VI

Primary cultures of retinal pigment epithelial (RPE) cells from cats with mucopolysaccharidosis VI (MPS VI) have been initiated from mixed populations of cells (ie, derived from the entire eyecup and represented by both pigmented and nonpigmented RPE cells). The cells were enzymatically dissociated from the eyecup and seeded at 6 X 10(4) cells/cm2. Cells from normal and affected cats formed confluent monolayers of polygonal cells between 5-10 days in culture and maintained most of their in vivo morphologic characteristics. The only abnormality observed in the MPS VI-affected cultures was the accumulation of vacuolated intracytoplasmic inclusions; when numerous, these vacuoles caused cellular hypertrophy. Hypertrophy was present only in cells devoid of pigment. Pigmented cells adjacent to or near the hypertrophied cells exhibited little or no accumulation of vacuoles. The inclusions were indistinguishable from those observed in vivo in terms of size, distribution, and appearance. The MPS VI-affected RPE exhibited deficient arylsulfatase B (ASB) activity (RPE-ASB activity: normal = 506 nmol/hr/mg protein; affected = 22 nmol/hr/mg protein), whereas the activities of two other lysosomal enzymes, arylsulfatase A and alpha-L-iduronidase, were normal. A method was developed to initiate primary cultures of RPE cells from defined regions of normal and MPS VI-affected eyes. Studies indicated that cultures initiated from superior-equatorial regions (RPE nonpigmented) contained more vacuolated cytoplasmic inclusion than those initiated from inferior-equatorial regions (RPE pigmented). These findings indicated that the spatial distribution characteristic of the disease in vivo was maintained in culture and that disease expression was inversely correlated with pigmentation.     

Modulation of keratin and connexin expression in limbal epithelium expanded on denuded amniotic membrane with and without a 3T3 fibroblast feeder layer

PURPOSE. Based on the knowledge that limbal epithelial stem cells (SCs) do not express keratin-3 (K3), connexin (Cx)43, and Cx50, a study was conducted to investigate amniotic membrane (AM) culturing conditions that promote limbal SC expansion. METHODS. Human limbal epithelium was expanded on intact and epithelially denuded AM, with or without a 3T3 feeder layer, and subsequently transplanted to nude mice to induce epithelial stratification and differentiation. Immunostaining and Western blot analysis were used to determine protein expression of K3, Cx43, and Cx50. Expression of integrin-alpha3, -beta1, -alpha6, and -beta4 was investigated by immunostaining. RESULTS. Protein levels of K3, Cx43, and Cx50 in limbal epithelium on intact AM was lower than those on denuded AM. Addition of 3T3 to denuded AM increased the level of Cx43 but decreased that of Cx50. After xenotransplantation, the basal layer of the resultant stratified epithelium on intact AM did not express K3, Cx43, and Cx50, whereas that on denuded AM expressed all three markers. The addition of 3T3 resulted in positive staining of Cx43 and K3 but negative staining of Cx50 in the basal epithelium. After stratification, integrin expression was detected at the basal epithelium-amniotic basement membrane interface in all three culture conditions. CONCLUSIONS. Limbal cultures on intact AM retain a limbal epithelial phenotype, whereas those on denuded AM differentiate into a corneal phenotype. The addition of 3T3 slows but does not prevent corneal differentiation on denuded AM. Such a difference may involve integrin-mediated extracellular matrix interactions.     

Regulation of HLA class II antigen expression on cultured corneal epithelium by interferon-gamma.

The effect of recombinant human interferon gamma (IFN-gamma) on the induction of HLA class II (HLA-DR, -DP, -DQ) antigen expression on human corneal epithelial (HCE) cells was examined in different stages of culture. Primary cultures were established with limbal explants without endothelium. HCE cells in Stage 1 and Stage 2, with cells negative and positive for the 64K corneal keratin (the marker for advanced corneal epithelial differentiation), respectively, were prepared. HCE cells in both stages were treated with IFN-gamma at a concentration of 0 to 1000 U/ml for two to six days and were stained by the avidin-biotin peroxidase complex method. Class II antigens were not detected on HCE cells in either stage without IFN-gamma treatment. IFN-gamma induced three class II antigens on HCE cells in both stages in a dose- and time-dependent manner but at different levels for each antigen (DR greater than DP greater than DQ). In addition, DQ expression was related to cell differentiation, with DQ extremely rare at Stage 1 and more frequent at Stage 2 (5% vs. 20%). These findings indicate that the induction of class II antigens on HCE cells may be regulated by IFN-gamma independently for each of the antigens and that DQ induction may depend upon the differentiation of HCE cells in culture.     

Corneal epithelial wound healing in the absence of limbal epithelium.

Corneal epithelial stem cells are thought to be at the limbus. The limbal epithelium was surgically removed in 12 New Zealand white rabbits. After 6 months, four showed mild vascularization. To challenge the remaining proliferative reserve, two consecutive 7.5-mm epithelial woundings were created 3 weeks apart in 11 limbal-deficient corneas and 11 controls. After the first wounding, five of the limbal-deficient corneas showed delayed healing, and seven became moderately vascularized; the controls healed normally. After the second wounding, eight experimental corneas showed intense vascularization; the controls did not. Recurrent erosions with delays in healing were noted in nine experimental animals but not in controls. Flat-mount preparation and impression cytology revealed centripetal migration of conjunctival epithelium with goblet cells onto the experimental corneas. These results indicate that only limited proliferative capacity of corneal epithelium remains in the absence of limbus. The constellation of delayed healing with recurrent erosion, corneal vascularization, and conjunctival epithelial ingrowth can be considered possible signs of limbal stem cell dysfunction.     

Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane.

PURPOSE: Stem cell (SC)-containing limbal basal epithelium and transient amplifying cell (TAC)-containing corneal basal epithelium lie on different mesenchymal matrices. The gap junction protein connexin 43 (Cx43) is absent in the limbal basal epithelium but is present in the corneal basal epithelium, suggesting that the expression of Cx43 denotes SC differentiation into TACs. Amniotic membrane (AM) can expand limbal epithelial progenitor cells in vivo and in culture for subsequent corneal surface reconstruction. In this study, the modulation of Cx43 expression, gap junction intercellular communication (GJIC), and proliferative activity of ex vivo expanded human limbal epithelial (HLE) cells on intact and epithelially denuded AM was investigated. METHODS: HLE cells were expanded on intact (i.e., remaining devitalized amniotic epithelium) or epithelially denuded AM (EDTA-treated). Cx43 expression and 24-hour 5-bromo-2'-deoxyuridine-5'monophosphate (BrdU) labeling index (percentage) were determined by double immunostaining. GJIC was investigated by a scrape-loading dye transfer assay. In a subset of cultures Cx43 and K3 keratin as well as BrdU-retaining nuclei were analyzed in the stratified epithelium obtained 5 days after subcutaneous transplantation in NIH bg-nu-xidBR mice of AM cultures continuously labeled with BrdU for 7 days. RESULTS: The outgrowth rate, overall, was significantly higher on EDTA-treated AM than on intact AM (P < 0.05). Cx43 was expressed in 12.4% +/- 14.5% (n = 5) on intact and 57.5% +/- 18.2% (n = 5) on EDTA-treated AM (P < 0.05). The BrdU labeling index was 2.4% +/- 0.9% (n = 5) for the intact AM group, which was significantly less than 22.5% +/- 8.2% (n = 5) for EDTA-treated AM (P < 0.05). BrdU-labeled cells did not express Cx43. The dye transfer assay revealed reduced GJIC on both AM-cultured groups compared with the control culture on plastic (P < 0.002). GJIC on intact AM (17%) was reduced compared with that on EDTA-treated AM (27%; P = 0.42). After xenotransplantation, the basal layer of the stratified epithelium was Cx43 and K3 keratin negative and retained BrdU on intact AM, resembling characteristics of the limbal basal epithelium in vivo. In contrast, that of EDTA-treated AM was Cx43 and K3 keratin positive without BrdU retention, resembling characteristics of the corneal epithelium in vivo. CONCLUSION: These data indicate that denudation of the devitalized amniotic epithelium to expose its basement membrane might be a microenvironmental cue to promote TAC differentiation. The model system described herein is ideal for future exploration of the exact mechanistic operation in the microenvironmental niche that maintains the "stemness" of limbal SCs as well as in the signal that promotes corneal TAC differentiation.     

体外人羊膜培养兔角膜缘上皮细胞的细胞形态与连接复合体形成的研究

目的观察体外人羊膜上培养出的兔角膜缘上皮细胞形态特征及与羊膜基底膜连接复合体的形成。方法将15只新西兰白兔角膜缘组织（2mm×2mm）,于37℃ 5％ CO2孵箱中用1．2U／ml裂解酶Ⅱ处理20min,之后在人羊膜上皮面先行浸液培养2周,后行气-液培养2周。于培养期间,每周定期进行透射电镜检查,以观察连接复合体的形成过程,培养至第4周后,采用AE5免疫组织化学、高碘酸．希夫、苏木精-伊红等染色方法鉴定分化的上皮细胞,鉴别杯状细胞,观察细胞的形态特征。结果兔角膜缘上皮细胞体外培养10—14d可充满人羊膜面,对AE5免疫组织化学方法染色分化出的上皮细胞均呈阳性而高碘酸．希夫染色均呈阴性。苏木精-伊红染色显示从兔角膜缘上皮细胞分化出的上皮细胞与正常兔角膜上皮细胞相比无明显差异;自培养至第14天均未见与羊膜基底膜的连接复合体形成;于第21天时仅观察到初始阶段的连接复合体,第28天时仍未见明显变化。结论于体外人羊膜上培养的兔角膜缘上皮细胞分化出的细胞是角膜上皮细胞,其形态与正常兔角膜上皮细胞相同,并与羊膜基底膜仅形成初始阶段的连接复合体。     

The role of NGF signaling in human limbal epithelium expanded by amniotic membrane culture

PURPOSE: Amniotic membrane (AM) transplantation facilitates rapid epithelialization in severe neurotrophic corneal ulcers. To elucidate its action mechanism, we investigated the expression of ligands and receptors of the neurotrophin family by human limbal epithelial (HLE) cells expanded on AM cultures. METHODS: Expression of nerve growth factor (NGF); neurotrophins (NT)3 and NT4; brain-derived neurotrophic factor (BDNF); tyrosine kinase-transducing receptors TrkA, TrkB, and TrkC; and a pan-NT low-affinity receptor (p75(NTR)) was examined by immunostaining in the normal human corneolimbus, HLE grown on intact epithelially denuded AM, and stratified HLE, after subcutaneous implantation in NIH-bg-nu-xid BR mice. NGF protein level was assayed by an ELISA in extracts of intact and epithelially denuded AM. K252a, a specific inhibitor of TrkA autophosphorylation, was added to test whether it would inhibit HLE expansion on AM culture. RESULTS: Strong positive TrkA staining was confined to the basal epithelial cell layer of normal corneal and limbal epithelia, with the highest intensity noted in the limbus. TrkA staining was also strongly positive in the basal layer of HLE cells cultured on intact and epithelially denuded AM and in basal and some suprabasal layers of stratified HLE transplanted in nude mice. Positive staining of p75(NTR) was noted in the full-thickness of the corneal epithelium but was limited to the superficial layers of the limbus and in HLE cells cultured on intact and epithelially denuded AM, but was weak in HLE transplanted to nude mice. Weak staining of NT3 and TrkC was noted in the suprabasal layers of corneal and limbal epithelia but was negative in the stratified HLE in nude mice. Negative staining of NGF, NT4, BDNF, and TrkB was noted in all specimens tested. The NGF protein level was readily measured as 35.6 +/- 9.1 and 41 +/- 12.5 pg/mg protein in the homogenate of the intact and epithelially denuded AM, respectively (P = 0.0256). K252a significantly inhibited the HLE outgrowth on intact AM culture (P = 0.024). CONCLUSIONS: The strong expression of TrkA but not p75(NTR) in the limbal basal epithelial cells in vivo suggests that NGF signaling favors limbal epithelial stem cell survival. Such a phenotype is preserved in HLE cells on AM. Blocking NGF signaling significantly retarded HLE expansion on AM, supporting the notion that NGF is important in expansion of limbal epithelial progenitor cells. Furthermore, a high and therapeutic level of NGF was present in AM. Collectively, these findings indicate that denervated neurotrophic ulcers are associated with poor epithelial stem cell function at the limbus. Future studies are needed to determine whether AM transplantation to heal such ulcers may include the promotion of nerve regeneration and survival of epithelial progenitor cells.     

The presence of keratin 5 as an IgG Fc binding protein in human corneal epithelium

Rabbit immunoglobulin G (IgG) binds specifically to the human corneal epithelium. We investigated this phenomenon to identify the binding material in the tissue. Sections of human cornea were immunostained with negative-control grade rabbit IgG, F(ab')(2) (the antigen recognition fragment of IgG yielded by pepsin digestion), and Fc fragments. Human corneal epithelium was homogenized in a buffer containing Triton X-100 and the water-insoluble residue was subjected to two-dimensional gel electrophoresis. Western blotting was performed on the gels to detect materials that bound with rabbit IgG Fc fragments. Spots in the gel that bound with the Fc fragments were collected and analysed by peptide mass fingerprinting (PMF). Using an antibody against a binding candidate, keratin 5 (suggested by PMF analysis), we checked the occurrence of this phenomenon. Immunohistochemistry showed that rabbit IgG bound to corneal epithelium via the Fc region but not F(ab')(2). Western blots using rabbit Fc fragments showed that some protein spots in the gel bound to the fragments. All of the binding materials were basic and had molecular weights of approximately 55 kDa. PMF analysis showed that the binding material was keratin 5; all binding materials on the transferred membrane were recognized by an anti-keratin 5 antibody. On corneal sections, anti-keratin 5 produced staining similar to that of rabbit IgG. These findings suggest that rabbit IgG binds to keratin 5 in the human corneal epithelium via the Fc region of the molecule.     

Rubidium transport in cultured monkey retinal pigment epithelium.

Rb+ influx was used to assess Na-K-Cl cotransport and Na,K-ATPase activities in cultured monkey retinal pigment epithelium. Bumetanide-sensitive (Na-K-Cl cotransport-mediated) Rb+ influx exceeds ouabain-sensitive (Na,K-ATPase-mediated) Rb+ influx, with these two transporters accounting for approximately 95% of total Rb+ uptake. Half-maximal inhibition of Rb+ influx by bumetanide is attained at 75 nM bumetanide. The bumetanide-sensitive Rb+ influx depends on both extracellular Na+ and Cl-, and is activated by extracellular Rb+ with a relatively high affinity. Na-K-Cl cotransport activity is stimulated (2.5-fold) by increased extracellular osmolarity. Elevated cAMP content and glycolytic inhibition both depress cotransport activity. Cyanide application, however, had very little effect on Na-K-Cl cotransport activity. Monkey retinal pigment epithelial cells, maintained in culture, provide a system in which the activity and regulation of cation transport mechanisms can be examined.