Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation.

The authors studied the distribution of specific keratins within the superior, inferior, medial, and lateral regions of human limbus and cornea to determine whether the limbal epithelium exhibits regional heterogeneity in its microstructure. A corneal epithelial basic keratin (K3), recognized by monoclonal antibody AE5, was immunohistochemically undetectable in the basal layers of the limbus in these four regions, but was seen in all layers in the central cornea. The pattern of immunostaining with another monoclonal antibody, AE1, which recognizes several acidic keratins, was complementary to AE5 staining in that AE1 recognized a similar heterogeneity in the limbal epithelial cells. AE1 immunoreacted with the basal cells of the limbus, but not those of the central corneal epithelium. Limbal characteristics, as defined by AE1-positive and AE5-negative staining, extended deeply into peripheral cornea in the superior and inferior regions, but to a lesser extent in the lateral and medial regions. The broader regions of epithelium with limbal characteristics in the superior and inferior regions raises the possibility that these regions play an important role in corneal epithelial maintenance and wound healing.     

Gross factors in treatment of nonhealing corneal ulcers and recurrent erosions

Epidermal growth factor (EGF) is known to promote corneal epithelial wound healing in experimental scrape and keratectomy models. We studied its efficacy in treating alkali-burned epithelial ulceration. EGF induced hyperplasia and cell proliferation to resurface the denuded and denatured corneal stroma, but it did not prevent recurrent erosions. A combination of EGF with fibronectin (Fn) was noted to enhance epithelial defect closure after alkali burns of the cornea and was seen to prevent recurrent erosions. Histopathologic examination of these corneas revealed marked leukocytic infiltration of the alkali-burned corneal stroma. To find ways to retard this inflammatory response, we studied the role of topical steroids and their efficacy when used with EGF, Fn, and laminin (Ln) in the management of alkali-burned corneas. Use of steroids decreased the incidence of recurrent erosions and corneal perforations. Histologically, steroids markedly decreased the leukocytic infiltration of stromal tissue, thereby retarding the collagenolysis. Topical steroids used with EGF and fibronectin were seen to promote epithelial wound closure and to prevent recurrent erosions in alkali-burned corneas. Combinations of EGF, fibronectin, and steroids may have a place in the treatment of clinical corneal alkali burns.     

Keratin filaments of corneal epithelial cells

Intermediate filaments with a diameter of approximately 8–10 nm are present in great abundance in the cytoplasm of corneal epithelial cells. In order to study the immunological relationship between these filaments and the tonofilaments of epidermal cells, we prepared an antiserum against keratin proteins isolated from the stratum corneum of human epidermis. Immunofluorescent staining of frozen sections of rabbit and human corneas with this anti-keratin antiserum resulted in the intense staining of the entire corneal epithelium, but no staining of the endothelium or any of the stromal components. Similar staining of cultured rabbit and human corneal epithelial cells revealed a cytoplasmic network of wavy fibers distinct from microfilaments and microtubles. Electron microscopic examination of cultured rabbit corneal epithelial cells showed that they formed a stratified squamous structure with prominent desmosomal cell-cell junctions and wavy bundles of cytoplasmic 8–10 nm filaments. These results suggest that the 8–10 nm filaments of corneal epithelium are immunologically related to tonofilaments of the epidermis and are composed of keratin proteins. Since corneal epithelium is the only keratin-containing cell type in normal cornea, anti-keratin staining provides a simple, specific and sensitive method for identifying corneal epithelial cells. Further characterization of the keratins synthesized by corneal epithelial cells under various in vivo and in vitro conditions may provide useful information concerning the mechanisms of corneal epithelial differentiation.     

角膜中央碱性烧伤上皮愈合的实验研究

目的:观察角膜中央碱性烧伤后角膜上皮的形态学变化。方法:用浸有0.5mol/L NaOH溶液的8mm直经圆形纸片放在39只兔角膜中央表面1分钟,制备兔角膜中央碱性烧伤模型,用荧光素染色裂隙灯检查、光镜、扫描电镜和透射电镜进行研究。结果:角膜烧伤后即出现上皮溶解脱落,伤后8小时烧伤边缘处上皮细胞呈梭形,向损伤区内伸出突起;24小时大部分区域已有单层上皮覆盖,第4天角膜上皮再脱落。修复的上皮表面微绒毛和桥粒结构少,细胞间隙明显增宽。结论:角膜中央碱性烧伤上皮愈合主要是通过自外周正常区上皮细胞向损伤区增生移行来完成。再生的角膜上皮细胞间连接结构的减少可能与上皮易脱落有关。眼科学报1999;15:74—77。     

Prevention of stromal ulceration in the alkali-burned rabbit cornea by glued-on contact lens. Evidence for the role of polymorphonuclear leukocytes in collagen degradation.

Stromal ulceration of the alkali-burned rabbit cornea was found to be associated invariably with phagocytically active polymorphonuclear leukocytes (PMNs). A glued-on methylmethacrylate lens applied to corneas soon after burning, however, prevented re-epithelialization and also prevented PMN infiltration of the stroma and stromal ulceration. Subsequent partial detachment or complete removal of the lens resulted in epithelial resurfacing of the stroma, PMN infiltration, and stromal ulceration. Glued-on lenses applied to already ulcerating corneas arrested further ulceration by prohibiting additional PMN infiltration. Either surface debridement or glued-on methylmethacrylate rings also prevented re-epithelialization and ulceration in stromas not infiltrated by PMNs, but neither treatment was sufficient to prevent ulceration in corneas already containing numerous PMNs. The data suggest the possibility that the epithelium stimulates infiltration of the stroma by PMNs which then participate in stromal matrix degradation. Although no claim is made that only PMNs mediate matrix destruction in corneal ulceration, the efficacy of the lens would seem to be due to exclusion of the epithelium and the consequent prevention of stromal infiltration by PMNs.     

Epidermal growth factor in alkali-burned corneal epithelial wound healing

We conducted a double-masked study to evaluate the effect of epidermal growth factor on epithelial wound healing and recurrent erosions in alkali-burned rabbit corneas. Epithelial wounds 10 mm in diameter healed completely under the influence of topical epidermal growth factor, whereas the control corneas did not resurface in the center. On reversal of treatment, the previously nonhealing epithelial defects healed when treated with topical epidermal growth factor eyedrops. Conversely, the epidermal growth factor-treated and resurfaced corneas developed epithelial defects when treatment was discontinued. Histopathologic examination disclosed hyperplastic epithelium growing over the damaged stroma laden with polymorphonuclear leukocytes when treated with epidermal growth factor eyedrops, but it did not adhere to the underlying tissue. Hydropic changes were seen intracellularly as well as between the epithelial cells and the stroma.     

Macroscopic observations on corneal epithelial wound healing in the rabbit

A newly-developed macroscope was applied to observe the healing process of corneal epithelial wound in vivo. After removing epithelium of the central cornea, the changes of the corneal surface were observed with the macroscope and the findings were compared with histological examinations. At 12 hours after abrasion, areas unstained with Richardson's staining (R staining) appeared. In the histological section, a single layer of regenerating epithelial cells covered the same area. At 24 and 36 hours after abrasion, the epithelial defects became smaller but surrounding epithelium was rough and showed dot-like staining with R solution. By 2 days, the epithelial defects disappeared. On macroscopic observation, the central corneal surface showed a pavement-like appearance. Histology revealed that the regenerating epithelium still consisted of one or two layers. At 3 days, dot-like stainings were present only in the center and the corneal surface appeared considerably smooth. Histology also showed that regenerating epithelium became columnar and multilayered, thereby suggesting stratification. By 7 days, the abraded corneal surface had recovered its smooth appearance. Histologic sections also demonstrated that the epithelium had regained its normal structure. Thus, using this macroscope, findings suggesting the process of epithelial migration and proliferation could be observed.     

Differentiation in cultured limbal epithelium as defined by keratin expression.

The authors investigated differentiation of cultured corneal and limbal epithelial cells by immunochemically evaluating the changes in the profiles of keratins recognized by two monoclonal antibodies: AE5, which recognizes K3, and AE1, which recognizes a group of acidic keratins including K16, which is present in the hyperproliferative cells. After 1 and 2 weeks in culture, the human epithelial cells did not react with AE5 but did react strongly with AE1. At 3 weeks, only suprabasal cells exhibited a moderate reactivity with AE5, whereas AE1 binding was seen in all of the cells. After 5 to 6 weeks in culture, all of the cells reacted moderately with AE5 and AE1. Treatment of 2-week-old limbally derived cultures with mitomycin C (mitosis inhibitor) did not inhibit subsequent K3 expression. Thus, K3 expression was associated with maturation or a later stage of differentiation that did not require an additional cell division. Unlike human epithelial cells, rabbit suprabasal epithelial cells expressed K3 (reactivity to AE5) after only ten days in culture. The epithelium derived from central human cornea lost K3 by 1 week in tissue culture but expressed keratin(s) recognized by AE1. Even after 4-6 weeks, cells derived from the central cornea did not become confluent and did not react with AE5. Thus, limbally derived human and rabbit epithelial cells undergo chronological changes in K3 expression similar to that seen in rabbit epithelial cells derived from central cornea. However, cultured human limbal epithelial cells take a significantly longer time to express K3 (a phenotypic characteristic of differentiated corneal epithelium) than do rabbit epithelial cells.     

几种抗炎滴眼液对兔角膜上皮损伤和愈合影响的实验研究

目的 探讨双氯芬酸钠、妥布霉素地塞米松、普拉洛芬、溴芬酸钠滴眼液频繁点眼对兔角膜上皮的副作用,并观察其常规使用对兔角膜上皮创伤愈合的影响。设计 实验性研究。研究对象80只新西兰大白兔。方法 将兔分为2组,每组40只,一组为频点组（点滴眼液每小时1次）;另一组行直径6 mm的角膜上皮刮除模型并予点滴眼液每日4次。每组兔再随机分为5组,每组8只,分别给予生理盐水、双氯芬酸钠、妥布霉素地塞米松、普拉洛芬、溴芬酸钠滴眼液。选择右眼为观察眼。在角膜上皮损伤前、损伤后12、24、48、72、96小时及第5、6、7天进行观察,并在观察结束后摘除眼球行组织病理学检查。主要指标 眼表刺激症状、角膜上皮损伤、愈合情况和角膜上皮厚度。结果 与生理盐水组比较,四种滴眼液频繁点滴兔眼均未出现明显刺激症状且未见上皮缺损形成,但荧光素染色均可见角膜上皮点染,以第3天明显,且双氯芬酸钠组角膜上皮损伤的积分（9分）明显高于对照组（0分）和溴芬酸钠组（1分）（P<0.05）;病理学检查显示点药5天后,妥布霉素地塞米松组角膜上皮细胞层数（3.67±0.52层）少于对照组（4.17±0.41层）（P<0.05）,其余各用药组与对照组的角膜上皮细胞层数未见明显差异。创伤后兔角膜上皮的平均修复时间在双氯芬酸钠组和妥布霉素地塞米松组分别为（75.0±27.0）小时和（75.0±8.5）小时,而生理盐水、普拉诺芬和溴酚酸钠组分别为（66.0±11.1）小时、（69.0±15.4）小时和 （66.0±11.1）小时,各组比较差异无统计学意义（P>0.05）,但角膜上皮愈合后妥布霉素地塞米松组（2.00±0.00层）和双氯芬酸钠组（2.50±0.55层）上皮细胞的层数少于对照组（5.00±0.00层）（P<0.05）。结论  频繁滴用抗炎滴眼液可导致兔角膜上皮细胞潜在的损伤;双氯芬酸钠和妥布霉素地塞米松滴眼液抑制兔角膜上皮创伤的愈合作用略强于普拉洛芬和溴芬酸钠。     

Corneal epithelium-specific mouse keratin K12 promoter

Keratins are structural proteins expressed by epithelial cells. Approximately 30 different keratin proteins have been identified, each with a specific expression pattern in different epithelial cells. The tissue-specific promoter of several keratin genes have been used to direct the expression of transgenes in animals. Keratin K12 and K3 are expressed in differentiated and stratified corneal epithelium, although the relative expression of each appears to vary between species. We targeted the mouse K12 keratin gene in order to develop a tissue-specific promoter that could be utilized to study the functions of genes of interest expressed in the corneal epithelium. Three 5' truncated fragments of the keratin K12 promoter (1.03, 0.71 and 0.25 Kb) showed higher functional and tissue-specific promoter activity in a human corneal epithelial cell line than other cell lines. The 0.25 Kb K12 promoter fragment was also active in cultured rabbit corneal epithelial cells. Thus, increased expression in corneal epithelial cells directed by fragments of the mouse K12 promoter extended across species lines. The paired box homeotic gene 6 (PAX-6), which is involved in controlling eye development, stimulated the activity of keratin K12 promoter.     

Alteration of epithelial paracellular permeability during corneal epithelial wound healing.

We studied the paracellular permeability to mannitol of corneas with epithelium of corneal, limbal, or conjunctival origin. Corneas with epithelial defects reepithelialized by corneal or limbal epithelium were nonvascularized; the corneal permeability was initially increased and returned to normal 3 days later. When epithelial defects extended beyond the limbus, they were healed by conjunctival epithelium. If corneas remained avascular or minimally vascularized, the conjunctiva-derived epithelium underwent a transdifferentiation process into a cornealike morphology in which the corneal permeability was initially increased upon complete reepithelialization, and gradually decreased to a level similar to that of normal cornea, 4 weeks after healing. However, when corneas became vascularized, the conjunctiva-derived epithelium retained its original phenotype, and corneal permeability remained increased throughout the 8-month period of study. The deranged barrier functions noted in the above vascularized cornea were demonstrated further by horseradish peroxidase tracer, which was found in the intercellular spaces of conjunctiva-derived epithelium of vascularized corneas but not in the avascular corneas with epithelia of corneal or limbal origin, or transdifferentiated conjunctival epithelium. To study further the effect of subsequent ocular surface trauma, conjunctival biopsy was performed on transdifferentiated avascular corneas 3 months after initial wounding. The biopsy resulted in extensive vascularization in three of eight previously nonvascularized corneas. Two weeks later, the corneal permeability was increased to a level similar to that of conjunctiva. These results indicate that corneal epithelial paracellular permeability correlates well with the status of the epithelial phenotype.     

Loss of fenamate-activated K+ current from epithelial cells during corneal wound healing.

PURPOSE: The corneal epithelium provides a barrier between the external environment and the cornea. It also serves as an ion transporting epithelium. Because of its proximity with the external environment, the corneal epithelium is frequently injured through physical or chemical insult. The purpose of this study was to determine whether corneal epithelial cell whole-cell currents change during corneal wound healing as the author of the present study has previously reported for corneal keratocytes and endothelial cells. METHODS: Rabbit corneal epithelial cells were injured by scraping, heptanol exposure, or freezing. The epithelium was allowed to heal for 12 to 74 hours. Cells were dissociated from corneas, and whole-cell currents were examined using the amphotericin-perforated-patch technique. RESULTS: Cells from the wounded corneal groups had significantly increased capacitance values, indicating increased surface area compared with that of control cells. As previously reported, the primary control whole-cell current was a fenamate-activated K+ current. An inwardly rectifying K+ current and a Cl- current were also observed. In epithelial cells from heptanol-wounded corneas, these conductances were generally unchanged. In cells from scrape- and freeze-wounded corneas, however, the fenamate-activated current was absent or significantly attenuated. CONCLUSIONS: As they do in corneal keratocytes and endothelial cells, K+ channels disappear during some models of corneal epithelial wound healing. In addition, cell capacitance, a measurement of cell surface area, increases. These results suggest that substantial K+ channel activity is not required for in vivo epithelial cell proliferation during corneal wound healing.     

Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth

PURPOSE: To determine the kinetics of corneal epithelial maturation during embryonic development and postnatal growth. METHODS: Expression patterns of keratin (K)12 and K14 were determined in mouse embryos (embryonic days [E]15.5-19.5), corneas of postnatal day (P)0 to 10 months, and healing corneas after epithelial debridement in P30 and P90 mice. The expression of alkaline phosphatase (AP) was determined during postnatal growth and healing of epithelial debridement of Krt12(Cre/Cre)/ZAP bitransgenic mice. RESULTS: During embryonic development, K12 expression by corneal peridermal epithelium commenced at E15.5. In the period from E15.5 to P10, the expression of K12 was restricted to the suprabasal and/or superficial cells of the corneal epithelium, whereas the K14 expression was restricted to the basal cells. After P30, K12 expression was sporadically detected in the basal corneal epithelium, and the number of K12-positive basal cells increased as the mice grew older. The number of K14-positive cells that coexpressed K12 increased with age and reached a plateau after P180. Healing of the debrided epithelium facilitated the increase in K14-positive cells that coexpressed K12. Many basal cells of Krt12(Cre/Cre)/ZAP mice remained undifferentiated and expressed LacZ at P15, and they then differentiated to express Cre, which leads to excision of LacZ and AP expression. CONCLUSIONS: In the mouse, the corneal epithelium does not become fully mature until 3 to 6 months after birth, in that a significant number of corneal basal epithelial cells of young mice (相似文献   

In vitro tissue engineering of lamellar cornea using human amniotic epithelial cells and rabbit cornea stroma

AIM:To reconstruct the lamellar cornea using human amniotic epithelial (HAE) cells and rabbit cornea stroma in vitro using tissue engineering technology.METHODS: Human amnia taken from uncomplicated caesarean sections were digested by collagenase to obtain HAE cells, and the cells were cultured to proliferate. Rabbit corneal epithelial cells were removed by n-heptanol to make lamellar matrix sheets. The second passage of HAE cells were cultured on the corneal stroma sheets for 1 or 2 days, then transferred to an air-liquid interface environment to culture for 2 weeks. Tissue engineered lamellar cornea (TELC) morphology was observed by Hematoxylin-eosin (HE) staining; its ultrastructure was observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM); corneal epithelial cell-specific keratin 3 and keratin 12 were detected with immunofluorescence microscopy.RESULTS:HAE cells grew on the rabbit corneal stroma, forming a monolayer after 1-2 days. About 4-5 layers of epithelial cells developed after 2 weeks of air-liquid interface cultivation, a result similar to normal corneal epithelium. Rabbit corneal stromal cells were significantly reduced after one week, then almost completely disappeared after 2 weeks. TEM showed desmosomes between the epithelial cells; hemidesmosomes formed between the epithelial cells and the basement membrane. SEM revealed that the HAE cells which grew on the lamellar cornea had abundant microvilli. The tissue-engineered cornea expressed keratin 3 and keratin 12, as detected by immunofluorescence assay.CONCLUSION: Functional tissue-engineered lamellar corneal grafts can be constructed in vitro using HAE cells and rabbit corneal stroma.     

Effects of the Matrix Metalloproteinase Inhibitor GM6001 on the Destruction and Alteration of Epithelial Basement Membrane During the Healing of Post-alkali Burn in Rabbit Cornea

Purpose

To examine the alteration in structure and matrix composition of epithelial basement membrane (BM) during the healing of alkali-burned rabbit cornea, and the roles of matrix metalloproteinases (MMPs) in these alterations.

Methods

The central cornea of one eye of 78 albino rabbits was exposed to 1?N NaOH for 180?s under general and topical anesthesia and allowed to heal with or without subconjunctival injection of GM6001 (an MMP inhibitor). Cryosections of affected corneas were observed by H&;E staining, immunohistochemistry for type IV collagen subtypes, or in situ zymography for detection of localization of MMP activity.

Results

Uninjured corneal epithelial BM exhibited α5 (IV)-immunoreactivity, but lacked the α1/α2-immunoreactivity of collagen IV. Epithelial BM in healing burned cornea transiently exhibited α1/α2-immunoreactivity. Examination by in situ zymography showed an upregulation of MMP activity in the regenerated central epithelium and anterior stroma of the burned corneas at days 7 and 14. GM6001 suppressed degradation of α5-containing epithelial BM in vivo and also in organ culture.

Conclusions

Epithelial BM was degraded by endogenous MMPs during healing following an alkali burn in rabbit cornea. GM6001 had an inhibitory effect on the degradation of the epithelial basement membrane in burned cornea in vivo.?Jpn J Ophthalmol 2006;50:90–95 © Japanese Ophthalmological Society 2006     

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。     

Differentiation-dependent increases in lactate dehydrogenase activity and isoenzyme expression in rabbit corneal epithelial cells

Lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities were studied during corneal epithelial growth and differentiation in cell culture. LDH and G-6-PDH activities increased up to 60 and 150-fold, respectively, when corneal epithelial cells constituted a differentiated four to five layered epithelium; these increases showed a similar time-course to the expression of K3 keratin. Immunostaining experiments showed that in growing colonies, LDH staining is stronger in those cells that are K3 positive; in contrast, in confluent four to five layered epithelia LDH and K3 were located in all cell layers, similar to the pattern found in frozen sections from rabbit central cornea. During growth and differentiation, the LDH isoenzyme set from corneal epithelial cells did not change; and it was different from those observed in cultured conjunctival, esophageal and epidermal cells. The augment in LDH activity was due to a 25-fold increase in the LDH-H mRNA and a 12-fold augment in LDH-M mRNA. A computer-assisted search led to identify AP2 and Sp1 binding sites in the LDH and G-6-PDH promoters, suggesting that their expression might share common regulatory mechanisms with the regulation of the differentiation-linked keratins. It is proposed that LDH may be an early marker of corneal epithelial differentiation, and its isozyme pattern could be distinctive from other epithelial cell lineages.     

Actin filament localization in developing and pathologic human corneas

Actin is associated with motility, cell morphology, and cell-substrate adhesion. The molecular probe NBD phallacidin, which reacts with filamentous actin, was used to study the distribution of actin filaments in the corneal and conjunctival epithelium, stroma, and endothelium. Frozen sections of human fetal eyes from 8 weeks to 40 weeks of gestation were reacted with NBD phallacidin. Pathologic tissues included keratoplasty specimens from patients with hereditary posterior polymorphous corneal dystrophy (PPMD) and surgically excised tissues removed for treatment of epithelial down-growth. Normal human cornea was used as a control. Immunofluorescent staining disclosed actin filament distribution in corneal epithelium as early as 9-10 weeks of gestation. Staining increased with maturation until term. Adult human corneal epithelium showed more pronounced staining of the surface layers. Stromal staining was more extensive in earlier stages of gestation and decreased in later stages of gestation, after 20-21 weeks. In pathologic corneas with posterior polymorphous dystrophy, there was localization of actin, as well as keratin, in the abnormal epithelial-like layers lining the posterior cornea. In epithelial downgrowth, actin and keratin were demonstrated in multilayered squamous epithelium on the anterior iris surface. Actin appears to be involved in migration of corneal epithelial and endothelial cells.     

Expression of the 55-kD/64-kD corneal keratins in ocular surface epithelium.

According to the concept of keratin pairing defined by tissue coexpression, a 55-kD/64-kD keratin pair is a marker of "corneal-type" differentiation. Intermediate filament (IF)-enriched preparations from guinea pig and bovine corneal epithelium were analyzed, and a rabbit antiserum was generated against a 55-kD polypeptide enriched in these preparations. This antiserum generated a typical IF-like pattern in cultured bovine corneal epithelial cells. Immunofluorescence microscopic analysis of frozen sections of guinea pig and bovine tissue revealed that the 55-kD antiserum labeled corneal and limbal epithelium. In addition, the antiserum stained a subpopulation of peripheral limbal cells that were distributed in both basal and suprabasal layers of the epithelium. The monoclonal antibody AE5 was used to investigate the distribution of the 64-kD polypeptide in guinea pig and bovine tissue. Immunoblotting analysis revealed that AE5 antibodies recognized a 64-kD polypeptide in guinea pig cornea, but recognized a 66-kD polypeptide in bovine cornea. Immunofluorescence microscopic analysis of guinea pig tissue revealed that AE5 antibodies labeled suprabasal layers of corneal epithelium, in suprabasal layers of limbal epithelium, and in groups of cells in the peripheral limbal epithelium. We discuss the possibility that the ocular epithelial cells recognized by either the 55-kD or the 64-kD antibodies in the peripheral limbus may play a role in the reepithelialization of the cornea after wounding.     

角膜上皮干细胞缺失动物模型的方法研究

目的:建立兔角膜上皮干细胞缺失动物模型,为人工生物角膜上皮实验研究奠定基础。方法:方法一,单纯去除角膜缘浅板层环周内外宽各2mm,深约100～150μm角膜组织;方法二,在方法一的基础上,再去除角膜中央浅板层组织约100～150μm。术后,裂隙灯显微镜下观察,以角膜混浊、上皮荧光素染色及新生血管为参数评价,3项参数得分之和为该动物时间点总评分。以角膜新生血管评分在3分以上,角膜上皮混浊在2分以上,采用印迹细胞法角膜面取材行角蛋白K3单克隆抗体间接免疫荧光法检测表达为阴性的。结果:单纯去除角膜缘的动物模型10眼有4眼成功(40%),从制作至成功平均时间29.5±1.3d。角膜缘联合角膜中央板层组织共同去除组,10眼均成功(100%),从制作至成功平均时间为15.8±0.8d,两组成功率对比差异有统计学意义(χ2=8.58,P<0.01);成功天数比较差异有统计学意义(t=24.5,P<0.01)。两组成功模型角膜采用印迹细胞法取材行角蛋白K3单克隆抗体间接免疫荧光法检测表达均为阴性。结论:采用角膜缘联合角膜中央上皮共同去除法制备角膜上皮干细胞缺乏模型成功率高,周期短,值得推广应用。