TNF-α及其Ⅰ型受体(P55)在翼状胬肉中的表达

目的：检测不同翼状胬肉组织中肿瘤坏死因子（tumor necrosis factor alpha,TNF-α）及其Ⅰ型受体P55的表达变化,探讨TNF-α及P55在翼状胬肉发病机制中的作用。方法：免疫组织化学方法（PV）检测翼状胬肉（72眼）及胬肉旁结膜组织（30眼）中TNF-α及其受体P55的表达,分析与翼状胬肉临床病理特征的关系。结果：TNF-α在翼状胬肉和胬肉旁结膜组织中的阳性表达率分别为65.3%（47/72）,26.7%（8/30）,差异有显著统计学意义（χ2=12.706,P〈0.01）。P55在翼状胬肉和胬肉旁结膜组织中的阳性表达率分别为56.9%（41/72）及16.7%（5/30）,差异有显著意义（χ2=13.875,P〈0.01）。TNF-α在复发性胬肉的阳性表达率高于初发性胬肉（χ2=6.547,P=0.011）,表达强度差异比较无统计学意义（F=1.288,P=0.393）; 进展期胬肉阳性表达率高于静止期胬肉（χ2=4.082,P=0.043）,表达强度比较无统计学意义（F=0.489,P=0.708）。P55在复发性胬肉的阳性表达率高于初发性胬肉（χ2=9.907,P=0.002）,表达强度比较无统计学意义（F=1.175,P=0.424）; 进展期胬肉的阳性表达率明显高于静止期胬肉（χ2=11.140,P=0.001）,表达强度比较无统计学意义（F=0.665,P=0.621）。结论：TNF-α及P55在翼状胬肉中的阳性表达率随着发病状态和临床分期的进展而变化。TNF-α及P55表达与临床分类、临床分期、患者工作生存状况相关。TNF-α及P55在翼状胬肉不同发病状态及临床分期的表达强度无明显差异。     

翼状胬肉中Ki-67和VEGF的表达

目的了解Ki-67和VEGF在翼状胬肉组织中的表达。方法用免疫组织化学SP法对36例初发型翼状胬肉患者切除的翼状胬状组织进行Ki-67和VEGF的检测并与正常人结膜组织进行对照。结果Ki-67在所有的翼状胬肉组织上皮中均有表达,头部最为明显,全层均有不同程度的表达。在正常的结膜上皮内对Ki-67的表达较胬肉上皮明显减弱,仅在基底细胞层散在表达。VEGF在翼状胬肉上皮明显减弱,仅在基底细胞层散在表达。VEGF在翼状胬肉上皮基底细胞层细胞,血管内皮细胞以及成纤维细胞有阳性表达。结论翼状胬肉组织上皮中有Ki-67和VEGF的过度表达,提示翼状胬肉组织上皮中存在增殖调控的异常,上皮细胞可能发生了转化,上皮细胞在翼状胬肉的发生发展中可能起着关键的作用。     

VEGF、SDF-1、Ki-67、PCNA及Survivin与翼状胬肉的关系分析

目的：分析血管内皮生长因子(vascular endothelial growth factor,VEGF)、基质细胞衍生因子-1(stromal cell-derived factor 1,SDF-1)、肿瘤增殖抗原(Ki-67)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)及生存素(Survivin)与翼状胬肉的关系。

方法：选取2013-01/2015-05于本院进行诊治的79例106眼翼状胬肉患者为观察组,同时期的79例正常结膜者为对照组,然后将两组的组织VEGF、SDF-1、Ki-67、PCNA及Survivin阳性细胞数分级及染色强度分级进行比较,并比较其中不同性别、分期及分型患者的检测结果,同时以Logistic分析上述指标与翼状胬肉的关系。

结果：观察组的组织VEGF、SDF-1、Ki-67、PCNA及Survivin阳性细胞数分级及染色强度分级均高于对照组,不同分期及分型患者的检测结果也存在一定差异(均P<0.05),而不同性别患者间的检测结果则无明显差异(均P>0.05),经Logistic分析显示,上述组织指标均与翼状胬肉有密切的关系。

结论：翼状胬肉组织中的VEGF、SDF-1、Ki-67、PCNA及Survivin表达呈现异常状态,上述指标均与翼状胬肉有密切的关系。     

HIF-1与VEGF在翼状胬肉中的表达及意义

目的研究缺氧诱导因子-1（HIF-1）与血管内皮生长因子（VEGF）在翼状胬肉组织中的表达及其相关性。方法采用免疫组织化学法研究HIF-1与VEGF分别在32例翼状胬肉与11例人正常球结膜组织中的表达。结果32例翼状胬肉中HIF-1的阳性表达率为62.5%（20/32）,VEGF的阳性表达率为84.8%（27/32）,11例正常结膜组织中HIF-1的阳性表达率为9.1%（1/11）,VEGF的阳性表达率为18.2%（2/11）,正常球结膜与翼状胬肉组织中HIF-1与VEGF的表达差异有统计学意义,且翼状胬肉中HIF-1与VEGF的表达呈正相关（P〈0.05）。结论HIF-1及VEGF在翼状胬肉中高表达,提示其可能参与了翼状胬肉的发生和发展。HIF-1与VEGF在翼状胬肉中的表达呈正相关,提示在翼状胬肉中HIF-1可能参与了对VEGF的调控。     

血管生成拟态在翼状胬肉进展中的作用及可能机制

目的　研究血管生成拟态（VM）在翼状胬肉进展中的作用及其与基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-9（MMP-9）和血管内皮生长因子（VEGF）表达的关系,探讨VM与翼状胬肉进展的相关性及可能机制。方法　筛选符合条件的翼状胬肉标本,进行胬肉分期（静止期/进展期）、分型（初发型/复发型）及临床特征指标采集。采用CD31/PAS双重染色检测不同类型翼状胬肉中VM的形成差异。以初发型进展期翼状胬肉为研究对象,HE染色观察病理特征,免疫组织化学染色及Western blot检测MMP-2、MMP-9及VEGF的表达,分析VM与初发型进展期翼状胬肉临床病理特征及MMP-2、MMP-9、VEGF的相关性。结果　静止期翼状胬肉VM阳性率为15.38%,进展期翼状胬肉VM阳性率为68.50%,组间差异具有统计学意义（P=0.000）;初发型翼状胬肉VM阳性率为40.80%,复发型翼状胬肉VM阳性率为81.48%,组间差异亦具有统计学意义（P=0.000）。VM与进展期及复发型翼状胬肉均呈正相关（均为P<0.05)。VM阳性的初发型进展期翼状胬肉组织,其血管分级、上皮细胞层数、杯状细胞数、成纤维细胞数、血管数、炎症细胞数、纤维化程度、MMP-2、MMP-9及VEGF的表达均明显高于VM阴性者,而变性程度低于VM阴性者,差异均有统计学意义（均为P<0.05）。初发型进展期翼状胬肉VM与翼状胬肉血管分级、上皮细胞层数、杯状细胞数、成纤维细胞数、血管数、炎症细胞数、纤维化程度、MMP-2、MMP-9及VEGF的表达均呈正相关,与胬肉变性程度呈负相关（均为P<0.05)。结论　翼状胬肉组织中存在VM结构,可作为其血供途径之一,VM可促进翼状胬肉的进展,MMP-2、MMP-9及VEGF可能是翼状胬肉VM形成的分子机制。     

血管生成拟态与翼状胬肉进行期及静止期的相关性研究

目的：研究血管生成拟态与翼状胬肉发生发展的相关性,初步探讨其临床意义。
　　方法：选取20例正常眼的结膜组织及翼状胬肉静止期和进行期各50例标本行苏木素－伊红（ HE）染色、过碘酸雪夫（ PAS）染色、免疫组化CD34、VEGF染色及免疫组化CD31染色联合PAS双重染色观察翼状胬肉新生血管和血管生成拟态情况。
　　结果：CD34和VEGF在翼状胬肉组织中的表达均高于正常结膜组织（两者P＜0．05）,并且两者在进行期翼状胬肉组织中的表达均高于静止期（两者P＜0．05）。 PAS染色血管拟态区网眼间隔染成紫红色,进行期和静止期出现血管生成拟态的例数分别是38例和11例,差异具有统计学意义（P＜0．05）,相关性分析显示血管生成拟态与进行期翼状胬肉呈显著密切正相关（ Spearman’s相关系数r＝0．540＞0．5,P＝0．000）。
　　结论：翼状胬肉除了新生小血管形成增多外,血管生成拟态也是其血供方式之一,并且血管生成拟态与翼状胬肉的进展具有密切关系。     

Toll样受体及VEGF因子在不同类型翼状胬肉中的表达和意义

目的 比较Toll样受体(TLR2、TLR3、TLR4)及血管内皮生长因子(VEGF)在初发性和复发性翼状胬肉中的表达差异:设计 实验研究。研究对象 初发翼状胬肉组织36例,复发翼状胬肉组织24例,正常结膜组织20例。方法 免疫组化法检测TLR2、TLR3、TLR4、VEGF及肿瘤坏死因子(TNF)-α在正常结膜、初发及复发性翼状胬肉组织中的表达情况。分析VEGF与TLR2、TLR3、TLR4表达的相关性。主要指标 TLR2、TLR3、TLR4、VEGF及TNF-α的表达量、阳性表达率及相关系数。结果 TLR2、TLR3、TLR4在正常结膜组、初发性翼状胬肉和复发性翼状胬肉的表达量依次上调。复发性翼状胬肉的间质层中存在大量VEGF棕黄色颗粒,上皮层中也有表达。VEGF与TLR2、TLR3和TLR4在复发性翼状胬肉中表达呈正相关(r=0.512,P=0.001;r=0.671,P=0.001;r=0.482,P=0.001)。结论 复发性翼状胬肉中TLR2、TLR3、TLR4、VEGF因子的表达高于初发性翼状胬肉,提示免疫炎症和VEGF因子可能是翼状胬肉复发机制中的重要因子,自身免疫炎症...     

CD34在翼状胬肉中表达的意义

目的研究翼状胬肉组织中CD34的表达及意义。方法对50例手术切除的翼状胬肉标本行苏木精-伊红染色、过碘酸希夫染色，并进行形态学、免疫组织化学研究和免疫荧光共聚焦显微镜下观察CD34、波形蛋白（VIM）、血管内皮生长因子（VEGF）在翼状胬肉中的表达。结果在翼状胬肉标本中，CD34在血管内皮细胞、血管周细胞以及一些散在的星形、梭形细胞中表达阳性，而在血管壁上及成熟的胶原细胞上呈阴性表达；VIM阳性表达于大部分翼状胬肉组织中；组织中部分疏松排列的网状及裂隙样、管样区结构过碘酸希夫染色呈阳性的紫红色反应；网状区的梭形细胞VEGF阳性表达。结论翼状胬肉组织中CD34阳性细胞来源于间充质干细胞的分化。翼状胬肉除以血管发生和血管新生的方式生成血管外，血管拟态可能是另外一种供血途径。     

糖皮质激素对翼状胬肉中SDF-1表达的影响

目的：研究基质细胞衍生因子-1（stroma cell-derived factor-1, SDF-1）和血管内皮生长因子（ vascular endothelial growth factor,VEGF）在翼状胬肉组织中的表达情况,探讨糖皮质激素应用对SDF-1表达的影响。 方法：选取翼状胬肉患者48例,其中随机选取24例患者在术前给予0.2g/L氟米龙眼药水点眼1wk。采用免疫组化SP法,检测翼状胬肉和正常球结膜组织中SDF-1、VEGF的表达。并使用IPP软件定量测定SDF-1、VEGF的光密度值。 结果：SDF-1和VEGF在正常结膜组织中仅见上皮基底层细胞有阳性表达或不表达,翼状胬肉中上皮全层细胞和血管内皮细胞均有阳性表达,其表达水平有显著性差异。SDF-1和VEGF在翼状胬肉中的表达呈显著相关性（ r=0.5235,P〈0.01）。糖皮质激素应用后,SDF-1和 VEGF的表达水平降低,差异有统计学意义。 结论：SDF-1、VEGF与翼状胬肉的发生发展存在着重要的联系,糖皮质激素可能通过减少SDF-1和VEGF的表达来抑制新生血管的形成。     

单酰甘油酰基转移酶2（MOGAT2）在翼状胬肉组织中的表达及意义

目的　研究单酰甘油酰基转移酶2（monoacylglycerol O-acyltransferase 2,MOGAT2）在翼状胬肉组织中的表达及意义。方法　采用免疫组织化学染色的方法检测54例翼状胬肉组织标本（原发50例,复发4例）和18例正常结膜组织标本中MOGAT2的表达情况,采用半定量积分法分析免疫组织化学表达的强弱,并对结果进行比较及统计学分析,同时分析翼状胬肉组中MOGAT2的表达与年龄、性别等影响因素及临床病理分期之间的关系。结果　免疫组织化学染色结果显示,54例翼状胬肉组织中MOGAT2强阳性表达8例,阳性表达29例,弱阳性表达8例,阴性表达9例;18例正常结膜组织中MOGAT2强阳性表达1例,阳性表达6例,弱阳性表达3例,阴性表达8例。MOGAT2在翼状胬肉组和正常结膜组中的表达差异有统计学意义（Z=-2.403,P=0.016）。在翼状胬肉组中,不同年龄、性别MOGAT2的表达差异均无统计学意义（均为P>0.05）,MOGAT2的表达强度与文化程度、职业、收入、户外工作时是否防护、是否吸烟、饮酒、血糖、血压情况及初、复发均无显著相关性（均为P>0.05）,但与翼状胬肉的临床分期有相关性,进展期翼状胬肉组织中MOGAT2表达显著高于静止期翼状胬肉组织（P<0.05）。结论　MOGAT2在翼状胬肉组织中呈高表达,进展期中的表达强度显著高于静止期,其表达水平的升高可能与翼状胬肉的发生发展有显著相关性。     

Ki-67 Labeling Index as a Marker of Malignancy in Ocular Surface Neoplasms

Purpose To evaluate the relationships among histopathological type, clinical malignant grade, and Ki-67 labeling index (LI) in sebaceous gland carcinoma (SGC), conjunctival squamous cell carcinoma (SCC), and conjunctival intraepithelial neoplasia (CIN), with pterygium and normal conjunctiva as controls.Methods This retrospective study was conducted at the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan. We used tissue specimens obtained from 20 patients (four SGC, four SCC, four CIN, four pterygium, and four normal conjunctiva). Ki-67 immunohistochemical analysis was performed in all 20 cases.Results The Ki-67 labeling index (LI) was 46.1 ± 3.0% (average ± SD) in SGC, 28.4 ± 4.5% in SCC, 20.0 ± 7.2% in CIN, 9.0 ± 2.2% in pterygium, and 6.8 ± 2.3% in normal conjunctiva. Ki-67 LI was significantly (Mann-Whitney U test, P < 0.05) higher in SGC than in SCC, and higher, but not significantly, in SCC than in CIN. Ki-67 LI was significantly (P < 0.05) higher in SCC and CIN than in pterygium.Conclusions These results suggest that Ki-67 LI may be a sensitive marker for ocular malignant tumor grading. Jpn J Ophthalmol 2004;48:524–529 © Japanese Ophthalmological Society 2004     

Expression of p27(KIP1) and cyclin D1, and cell proliferation in human pterygium

BACKGROUND: The pterygium is a growth onto the cornea of fibrovascular tissue that is continuous with the conjunctiva, whereas the mechanisms of cell proliferation in pterygium epithelium are unknown. AIM: To analyse the histopathology and the expression of cell cycle-related molecules in pterygium tissues. METHODS: Seven pterygia were surgically removed using the bare-sclera procedure, and three normal bulbar conjunctivas were also obtained. Formalin-fixed, paraffin-wax-embedded tissues were analysed by immunohistochemistry with anti-p27(KIP1), cyclin D1 and Ki-67 antibodies. RESULTS: Conjunctival epithelium consisted of several layers of round cells with a few goblet cells. Nuclear immunoreactivity for p27(KIP1) was noted in many normal epithelial cells, where cyclin D1 and Ki-67-positive nuclei were intermingled. A variety of goblet cells were located in the superficial layer of the pterygium head as well as those of the body epithelia. Several pterygium epithelial cells were p27(KIP1) positive, whereas nuclear immunoreactivity for cyclin D1 and Ki-67 was detected in many epithelial cells. By contrast, immunoreactivity for p27(KIP1), cyclin D1 and Ki-67 was hardly detected in the pterygium stroma. CONCLUSION: It is suggested that pterygium growth and development are associated with the proliferation of epithelium, which is possibly involved in the expression of cell cycle-related molecules.     

Angiogenesis in pterygium: study of microvessel density, vascular endothelial growth factor, and thrombospondin-1

PURPOSE: This retrospective study aims to elucidate the role of angiogenesis in the pathogenesis of pterygium. We evaluated microvessel density (MVD), and expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). METHODS: Fifty-two surgically excised pterygia and seven normal conjunctivae were immunohistochemically studied applying the streptavidin-biotin method in paraffin-embedded tissue sections. Monoclonal antibodies were targeted against CD31, VEGF, and TSP-1 proteins. Results: Pterygium presented with statistically significant higher average count of microvessels compared to normal conjunctivae (17.97+/-8.5 vs5.72+/-5 per high power field, P=0.001). In 24/52 (46.2%) cases of pterygium, high expression levels for VEGF were demonstrated, whereas the mean percentage of VEGF-positive epithelial cells was 58.03%. Furthermore, normal conjunctival presented statistically significant higher expression levels for VEGF in epithelial cells (83.14+/-36.08 vs58.03+/-31.23%, P=0.007). On the contrary, the presence of VEGF immunoreactivity in vascular endothelial and stromal cells was significantly higher in pterygium tissues (P<0.0001). Stromal staining for TSP-1 was detected in only 29/52 (55.8%) of the cases and no correlation with normal conjunctivae was found. Finally, statistically significant positive correlation between MVD values and stromal VEGF expression was found (P=0.049). CONCLUSION: The angiogenesis-related factors that were studied proved to be highly expressed in pterygium tissue. On the contrary, TSP expression level was low, allowing inducers of angiogenesis to act uninhibited. This phenomenon could provide the pathogenic basis of pterygium formation.     

Expression of p63 and p16 in primary and recurrent pterygia

Background p63 and p16 have been described as stem-cell markers of squamous epithelium. In an attempt to obtain new insights into the pathogenesis of pterygium, this study aims to evaluate the relationship between p63 and p16 expression in primary and recurrent pterygia.Methods Samples of primary (n=56) and recurrent (n=14) pterygia and normal bulbar conjunctival tissue (n=11) were submitted to immunohistochemical study to evaluate the expression of p63 and p16 in these tissues.Results Most of the cells stained for p63 were located in the basal layer of the normal conjunctiva, in the lower two-thirds of the epithelium of primary pterygia, and throughout all epithelial layers of recurrent pterygia. In normal conjunctivae, p16 expression was rarely expressed. Primary and recurrent pterygium groups exhibited increased p16 expression, with cytoplasmic staining in the primary group, and cytoplasmic or nuclear staining in the recurrent group.Conclusion The overexpression of p63 and p16 observed in the present study reinforces likelihood of involvement of these genes in the pathogenesis of pterygium, perhaps related to the intense cellular turnover with substitution of superficial epithelial cells by less differentiated forms. This loss of normal cellular differentiation of the epithelial layers could explain the high rates of recurrence overall in the recurrent pterygia.Research supported by FAEPA and CAPES.     

Comparative evaluation of lymphatic vessels in primary versus recurrent pterygium

Purpose

To compare lymphangiogenesis in primary versus recurrent pterygium.

Methods

Tissues from 88 excised primary and 34 recurrent pterygia were evaluated, and tissues from 7 nasal epibulbar conjunctivae segments were used as controls. The lymph-vascular area (LVA), lymph-microvascular density (LMD), and lymph-vascular luminal diameter (LVL) were examined and compared between the primary and recurrent pterygia. In addition, the expression of VEGF-A and VEGF-C in the primary and recurrent pterygia were determined by ELISA and real-time PCR. The relationships between the mRNA level and LVA, LMD, and LVL were clarified.

Results

Although there was no significant difference in quantification of LVL between primary and recurrent pterygia, the quantification of LVA and LMD in recurrent pterygia dramatically increased in comparison with primary pterygia (both P-values <0.01). Compared with primary pterygia, the VEGF-A and VEGF-C mRNA levels were up-regulated significantly in recurrent pterygia (both P-values <0.05). There was a significant relationship between VEGF-C mRNA and LVA, LMD, and LVL, while VEGF-A mRNA was only closely correlated with LMD in recurrent pterygia.

Conclusions

Lymphangiogenesis develops in recurrent pterygium, for which transient up-regulation of VEGF-C might be responsible.     

Expression of p53 and Ki-67 proteins in patients with increasing severity and duration of pterygium

Purpose:Pterygium is a triangular fibrovascular subepithelial ingrowth of degenerative bulbar conjunctival tissue over the cornea. It is now considered to be a result of uncontrolled cellular proliferation as overexpression of p53 protein and Ki-67 nuclear protein was found in the epithelium. This study was done to find the expression of p53 and Ki-67 with the severity and duration of the pterygium to explain the etiopathogenesis.Methods:Data were analyzed from 43 Indian participants of all age groups. All patients were divided according to the severity of pterygium (mild, moderate, and severe groups) and according to the duration of pterygium (<4 years and >4 years). The samples were studied by immunohistochemistry by using antibodies against p53 and Ki-67 proteins considering >5% expression as significant.Results:Of 43 cases, p53 and Ki-67 expression were positive in 33 cases. In mild, moderate, and severe cases p53 positivity was 33.3%, 78.4%, 100%, respectively. P53 expression increased with duration, 79.3% positive in <4 years, and 92.9% positive in >4 years. With increasing severity of pterygium, mild, moderate, and severe cases, Ki-67 positivity was 66.7%, 78.37%, 66.7%, respectively. Ki-67 expression with duration, 79.3% positive in <4 years, and 85.7% positive in >4 years of the duration of pterygium with no statistical significance.Conclusion:Our study revealed that with increasing duration and severity of pterygium, p53 expression was observed to be increasing. Ki-67 expression increased with the duration of pterygium but not with the severity.     

iNOS和CD34与复发性翼状胬肉的关联

目的：研究分析诱导型一氧化碳合成酶(inducible nitric oxide synthase,iNOS)和血管内皮因子CD34与复发性翼状胬肉发病之间的联系。

方法：选取2012-03/2014-10本院收治的复发性翼状胬肉患者15例作为本次研究的实验组,另选取同期15例单纯性胬肉患者作为对照组。通过免疫组化Envision法对两组患者的手术标本中的iNOS与CD34检测,并计算微血管密度(microvessel density,MVD),比较两组患者的iNOS与CD34阳性表达率和MVD值,分析iNOS和CD34对复发性翼状胬肉的影响作用。

结果：对照组中15例单纯性胬肉患者iNOS阳性表达率为7%(1例),实验组中15例复发性翼状胬肉患者iNOS阳性表达率为87%(13例); 对照组CD34阳性表达率为13%(2例),实验组CD34阳性表达率为80%(12例)。iNos与CD34在复发性翼状胬肉患者中较结膜正常人群中的阳性表达率明显增高,且均存在显著性差异(P<0.01)。实验组MVD值为46.02±10.88,对照组MVD为23.48±5.68,实验组MVD值明显高于对照组且两组存在统计差异(P<0.01)。

结论：iNOS与CD34在复发性翼状胬肉患者中阳性显著表达率高,说明iNOS与CD34与复发性翼状胬肉疾病的发生、发展存在一定联系。