翼状胬肉中细胞增殖与凋亡相关基因蛋白的表达及其意义

目的 :探讨增殖细胞核抗原 (PCNA)和抑凋亡基因Bcl 2、凋亡基因bax产物在翼状胬肉发病机理中的作用。方法 :应用SP免疫组织化学方法 ,检测 18例翼状胬肉、 9例正常结膜组织中PCNA、Bcl 2、bax基因产物的表达。结果 :PCNA在翼状胬肉中的阳性表达率显著高于正常结膜 (P <0 0 1) ,Bcl 2主要表达于翼状胬肉的上皮基底层细胞 ,正常结膜无表达 ,bax在翼状胬肉与正常结膜中的表达无显著差异。PCNA与Bcl 2表达呈正相关。结论 :翼状胬肉的发生、发展与PCNA和Bcl 2、bax的异常表达有关 ,可能是细胞异常增殖和凋亡失衡的共同结果。     

Telomerase activity and p53 expression in pterygia

PURPOSE: To investigate tolomerase activity and p53 expression in pterygial tissue. METHODS: Pterygia tissue was obtained during excisional surgery fr om 35 eyes of 35 patients, and superior bulbar conjunctival tissue from the same eye was also sampled as control when possible. Fluorescence telomeric repeat amplification protocol was used to measure telomerase activity in whole pterygium samples from 9 cases and in the epithelium and stroma of pterygium from another 10 cases. p53 protein content was measured by enzyme-linked immunosorbent assay (ELISA) in tissues obtained from 7 eyes, as well as in epithelial cell suspensions collected by brush cytology in 8 eyes. Six samples were also analyzed for UV-specific mutations in the p53 gene by the single-strand conformation polymorphism technique and DNA sequencing. A conjunctival epithelial cell line was irradiated with sublethal levels of UV-B to investigate whether telomerase activity can be induced in vitro. RESULTS: In all, 63% of pterygia samples demonstrated telomerase activity, whereas all 10 paired conjunctival control samples were negative (P = 0.05, chi-square test). Of the 10 samples in which telomerase activity was measured separately in the epithelium and stroma of pterygia, 5 samples were positive in the epithelium, only 1 of which had activity in the stroma. Average telomerase activity in positive samples was 18.44 +/- 8.77 U/microg protein, compared with telomerase activity measured in a carcinoma in situ patient (33.73 U/microg), and in an immortalized conjunctival epithelial cell line (50.72 +/- 15.55 U/microg). Telomerase activity was not upregulated in this cell line by UV-B exposure. All 6 pterygia samples tested for p53 mutations did not reveal the UV-specific mutations in exons 5, 6, 7, or 8. No statistical significance was observed in the pterygium or conjunctiva p53 protein levels in epithelial cells collected by brush cytology, while p53 protein level was lower in pterygia when measured in whole tissue samples. CONCLUSIONS: Telomerase activity was detected in some pterygia, mostly in the epithelium. Pterygia was not associated with an increase in epithelial p53 protein content measured by ELISA.     

UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway

PURPOSE: Pterygia are common, frequently recurring ocular surface lesions characterized by tissue remodeling, cellular proliferation, angiogenesis, and inflammation. The increased incidence of pterygia in persons exposed to excessive solar radiation suggests that ultraviolet (UV) light may play a critical role in the pathogenesis of this disease. These investigations were focused on the expression of collagenase-1 (matrix metalloproteinase [MMP]-1) in pterygia and cultured pterygium epithelial cells, to determine whether the expression of this protease could be modified after exposure to UVB. METHODS: Pterygium, conjunctival, and limbal epithelial cells were subcultured and exposed to various amounts of UVB. The conditioned medium and RNA were harvested for analysis by gelatin zymography, Western blot analysis, ELISA, and RT-PCR. Furthermore, whole pterygium specimens were irradiated to determine secreted MMP-1 levels. RESULTS: Immunohistochemical analysis revealed enhanced MMP-1 expression in pterygia that corresponded precisely with p63-positive epithelial cells. In contrast, significantly less MMP-1 reactivity was found in normal conjunctiva, limbus, and cornea. A dose- and time-dependent increase in MMP-1 was observed when pterygium epithelial cells were exposed to UVB with no significant modulation of inhibitor activity. MMP-1 was not affected in irradiated normal conjunctival epithelial cells or in pterygium fibroblasts but was induced in limbal epithelial cells. Although the induction of MMP-1 after UVB was not mediated by an intermediate soluble factor, the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) intracellular pathway was involved. CONCLUSIONS: Collectively, these data support the hypothesis of the involvement of UV light and MMPs in the development of pterygia and may assist in devising new therapeutic approaches for the treatment and prevention of pterygia.     

Impression cytology of pterygium.

The purpose of this study was to study the morphology and cytokeratin expression in the epithelia of pterygia. Impression cytology and immunohistochemical staining with antikeratin antibodies were performed in 32 eyes of 16 patients with pterygia. TUNEL stain and electron microscopy were also performed in surgical specimens ofpterygium. Squamous metaplasia-like epithelial cells were found in all specimens of impression cytology, especially in the head part. These specimens had positive immunostaining by antipancytokeratin antibodies, but not by anti-K12 AK2 mAb. Goblet cells were found around the area of these abnormal epithelial cells. TUNEL-positive cells were found in the epithelia of the pterygial head, but not in the body of pterygia and normal conjunctiva. The expressional patterns of keratin by these epithelial cells ofpterygia are consistent with the notion that they are derived from conjunctival epithelium and mimic the process of squamous metaplasia.     

Pax6在翼状胬肉上皮鳞状化生中的异常表达

目的:观察翼状胬肉上皮细胞中Pax6的表达情况.方法:应用免疫组织化学染色法检测15例翼状胬肉上皮细胞Pax6、K10、K19以及MUC5AC的表达,并与正常结膜对比.结果:在正常结膜上皮,K19全层表达,Pax6在全层细胞核内表达,K10染色阴性,MUC5AC阳性细胞在上皮表皮层呈单个或簇状散在分布;而翼状胬肉组织Pax6、K19和MUC5AC表达下降甚至阴性表达,且比较差异有统计学意义,而K10在上皮的部分表层细胞中阳性表达.结论:Pax6基因在翼状胬肉上皮细胞表达下调,提示其上皮细胞发生鳞状上皮化生.     

Expression of Fas-Fas Ligand Antigens and Apoptotic Marker APO2.7 by the Human Conjunctival Epithelium. Positive Correlation with Class II HLA DR Expression in Inflammatory Ocular Surface Disorders

Fas antigen (CD95) is a membrane receptor that plays a major role in induction of apoptosis. In surface conjunctival epithelial cells the expressions of Fas, Fas ligand, the apoptotic marker APO2.7 and of HLA DR class II antigen, a membrane marker known to be expressed in inflammatory conditions were investigated. Impression cytology specimens were collected in 65 patients: 20 normal ones, 15 contact lens wearers, 20 receiving chronic topical antiglaucoma treatment and 10 with nonspecific chronic conjunctivitis. Cells were processed for flow cytometry, using monoclonal antibodies to Fas, Fas ligand, APO2.7, HLA DR antigens and a negative isotypic control. Percentages of positive cells were recorded and levels of fluorescence quantified using fluorescent beads at standardized fluorescence intensities. In addition, a human conjunctival cell line was incubated with anti-Fas stimulating antibodies in order to test Fas-induced apoptosis in vitro. Fas was found in all specimens in most of the conjunctival cells, but quantitation of levels of fluorescence showed a significantly higher expression in pathologic eyes than in normal ones. Fas ligand and APO2.7 were variably expressed by conjunctival cells, but in a significantly higher percentage of cells in pathological eyes than in normal ones. In these eyes a strong expression of HLA DR was also observed, whereas normal eyes showed lowest levels. Highly significant correlations were found between Fas, Fas ligand, APO2.7 and HLA DR levels. Anti-Fas antibodies in vitro induced strong apoptosis in epithelial cells as confirmed by APO2.7 expression and DAPI staining. This study confirms that conjunctival epithelial cells normally express Fas antigen, and more inconstantly its ligand, as do corneal ones or keratinocytes. Fluorescence quantitation by flow cytometry showed much higher expression in inflammatory eyes than in normal ones, and demonstrated a strong correlation between apoptotic and inflammatory pathways in the ocular surface.     

Accumulation of p53 protein in pterygia is not accompanied by TP53 gene mutation

Previously, we reported that pterygial epithelial cells show positive p53 staining by immunohistochemistry, and that they do not demonstrate apoptosis. We wished to determine whether the accumulation of p53 protein was caused by missense mutations in exons 5-8 of the TP53 gene, as is frequently the case in malignant tumours that contain high levels of abnormal p53. From 11 pterygia, epithelial cells were isolated by laser capture microdissection, or manually, in order to reduce the contribution of TP53 from normal cells. DNA from pterygial epithelial cells was amplified across exons 5-8 in 10 pterygia and across exons 5,7 and 8 in another pterygium. In 2 pterygia, all translated exons (2-11) were sequenced. No mutations were found, although normal polymorphisms in codon 72 were readily detected in 2 pterygia. RT-PCR was used to compare amounts of TP53 mRNA isolated from normal conjunctiva and pterygia from eight additional patients. We detected an approximate two-fold increase of TP53 RNA in pterygia compared to that in normal conjunctiva. Western blotting was used to compare amounts of p53 protein in pterygia and normal conjunctiva. Consistent with our previous immunohistochemical studies, amounts of p53 protein in pterygia, detected by the western blotting, were elevated compared to those detected in normal conjunctiva and corneal limbal epithelium. However, the TP53 gene in pterygia is not mutated, and therefore, the elevated levels of p53 protein must result from a different mechanism than that seen in malignant tumours containing TP53 missense mutations. The increased amount of p53 protein in pterygial cells does not cause apoptosis or block cell proliferation, suggesting that these normal p53 functions are inactivated in pterygia.     

Overexpression of p53 tumor suppressor gene in pterygia

PURPOSE: To assess p53 gene expression in pterygia with and without recurrence. The pathogenesis of pterygium has not yet been determined. The most widely recognized etiologic factor is ultraviolet radiation, which leads to degeneration of the conjunctiva. However, pterygium was recently found to have several tumor-like characteristics. The p53 gene is a common marker for neoplasia, and is known to control cell cycle, cell differentiation and apoptosis. In this study we examined the expression of the p53 gene in primary pterygia with and without recurrence, searching for the pathogenesis of this very common lesion and for a prognostic factor for recurrence. METHODS: Immunohistochemical staining using a monoclonal antibody to human p53 (DO-7) was performed on 13 consecutive patients with primary pterygia, four pterygia without recurrence and nine pterygia which recurred during a 12-month follow-up. As a control we used two specimens of normal conjunctiva. RESULTS: Seven of the 13 pterygia specimens (54%) were positive for abnormal p53 expression. There was no difference between the groups with and without recurrence. Two out of four pterygia (50%) without recurrence and five out of nine (55.5%) pterygia with recurrence were positive. No pathological staining was observed in the control specimens. CONCLUSIONS: In this study, abnormal p53 expression was found in pterygial epithelium, suggesting that pterygium could be a result of uncontrolled cell proliferation, and not as a degenerative lesion. There seems to be no connection between abnormal p53 expression and recurrence.     

基质金属蛋白酶在翼状胬肉的表达

目的:研究基质金属蛋白酶(matrix metalloproteinases,MMPs)在翼状胬肉中的分布和活性状态,探讨MMPs在翼状胬肉病理过程中的作用。方法:翼状胬肉切除术患者26例27只眼,静止期18只眼,进行期9只眼,正常人球结膜6例,免疫组织化学法和明胶酶谱法分析MMP-2和MMP-9的表达和活性水平。结果:翼状胬肉标本有MMP-2和MMP-9表达,MMB-2主要分布在基底上皮细胞层,MMP-9在炎症细胞和血管内皮细胞有阳性染色,翼状胬肉静止期MMP-2的活性水平[(235±18.17)扫描单位]和进展期[(221±22.53)扫描单位]比较差异无显著意义(P>0.05),MMP-9活性水平在静止期[(46±32.21)扫描单位]和进展期[(159±19.28)扫描单位]比较差异有显著性意义(P<0.05)。结论:翼状胬肉中MMP-2和MMP-9表达增强,MMP-2和MMP-9活性水平升高可能与翼状胬肉的发展有关。     

Proliferative activity and p53 expression in primary and recurrent pterygia

PURPOSE: To assess p53 expression and proliferative activity in primary and recurrent pterygia from the same eyes. DESIGN: Retrospective comparative human tissue study. PARTICIPANTS: Tissue from excised primary pterygia that did not recur (group A, n = 10) was compared with tissue from primary pterygia that recurred (group B, n = 10) and to the recurrent pterygia tissue that was excised from subjects in group B (group C, n = 10). Ten normal conjunctivas served as controls (group D). METHODS: Sections from each pterygium were immunostained with the MIB-1 and bp53. 12 monoclonal antibodies that react with Ki-67 and p53 antigens, respectively. MAIN OUTCOME MEASURES: Proliferative activity was calculated as the mean of the MIB-1 positive cell count per eyepiece grid in high magnification (x40) (positive cell count/grid). Percentage of positive cells of all cells in the grid area was evaluated in the p53-stained sections. RESULTS: Proliferative activity was found in the epithelium overlying the pterygia and normal conjunctiva. The mean MIB-1 positive cell count/grid +/- standard error was 2.84 +/- 1.07, 1.74 +/- 0.82, 3.83 +/- 1.35, and 0.86 +/- 0.33 in groups A, B, C, and D, respectively (P = 0.17, Kruskal-Wallis). P53 staining was found in 50% of pterygia in groups A, B, and C; none of the normal conjunctival tissues showed p53 immunoreactivity. Four of five p53-positive tissues in group B were p53-negative in group C. In the p53-positive pterygia, less than 10% of cells were p53 positive. However, p53-positive pterygia had higher mean MIB-1 positive cell count/grid +/- standard error as compared with the p53-negative lesions, 4.56 +/- 0.94 vs 1.39 +/- 0.59 (P = 0.021, Mann-Whitney). CONCLUSIONS: p53 immunoreactivity and high proliferative activity in the epithelium overlying the pterygium are not associated with recurrence of pterygium.