角化细胞生长因子在中耳胆脂瘤的表达及意义

目的研究角化细胞生长因子（keratinocyte growth factor,KGF）在中耳胆脂瘤中的表达,探讨KGF对胆脂瘤上皮增殖能力的影响及其在中耳胆脂瘤发生、发展中的作用。方法20例胆脂瘤上皮标本及配对的正常外耳道皮肤标本制成石蜡切片,应用免疫组化SP染色法检测上述两组标本中KGF和Ki67的表达,对染色结果进行定量分析。结果KGF在胆脂瘤上皮中呈强阳性表达,且从基底层向角质层染色有逐渐增强的趋势,间质中可见散在的阳性细胞;正常外耳道皮肤标本主要表现为间质中稀疏不均的弱阳性表达,在上皮细胞不表达。胆脂瘤上皮和正常外耳道皮肤的KGF阳性表达率之间差异有显著性意义（P〈0.01）。在胆脂瘤上皮中KGF与Ki67表达呈正相关（r=0.609,P〈0.01）。结论KGF和Ki67表达与中耳胆脂瘤的增殖能力有高度相关性,局部炎症通过调控KGF的表达促进胆脂瘤上皮的异常增殖,其中KGF的自分泌机制可能与胆脂瘤的发生、发展密切相关。     

应用单克隆抗体Ki-67鉴定中耳胆脂瘤角化细胞的增生性

胆脂瘤是常见的中耳病患，据文献报道胆脂瘤上皮有异常增生活性。Ki67是一种通过细胞表达在细胞增殖周期的民，S，SZ和M阶段能识别人细胞核抗原的单克隆抗体，并能确定标本组织中的细胞增生活性。该作者运用Ki67表达来研究胆脂瘤和正常人皮肤标本，试图证实胆胀瘤上皮的高度增生活性。取鼓室成形术后的胆脂瘤和正常外耳道上皮标本各15份作低温保存，采用活性Ki－67抗体浓度为l：100，运用碱性磷酸酶抗碱性磷酸酶的免疫组化方法。结果显示：局限在正常外耳道上皮基底细胞层的角化细胞，有很少一部分细胞核被Ki67着色，而胆脂瘤上皮基底层…     

磷酸化酪氨酸在中耳胆脂瘤的观察

目的:探讨磷酸化酪氨酸在中耳继发性胆脂瘤的表达情况,分析其在胆脂瘤上皮增殖演变过程中的作用。方法:应用免疫组化ＳＰ染色方法和计算机图像分析系统,连续观察１０例具有典型鼓膜松弛部后皱襞处穿孔的中耳胆脂瘤患者的不同部位(胆脂瘤上皮、鼓膜穿孔部位邻近皮肤和耳道深部正常皮肤)中磷酸化酪氢酸的表达情况。结果:磷酸化酪氨酸在胆脂瘤上皮各层细胞均呈高度表达,以基底层和棘层最为明显;穿孔部位邻近皮肤则在基底层和棘层细胞中呈中等表达;耳道深部正常皮肤仅基底层细胞呈弱表达。各组之间差异均具有高度显著性意义(Ｐ＜０．００１)。结论:磷酸化酪氨酸在中耳继发性胆脂瘤中不同部位的表达呈连续性阶梯性上升,在胆脂瘤上皮中的高表达,说明胆脂瘤上皮具有高度增殖能力。在穿孔部位邻近皮肤中的中度表达,说明该处皮肤增生较活跃。     

Ras 蛋白在中耳胆脂瘤上皮中的表达

目的探讨Ｒａｓ蛋白在中耳继发性胆脂瘤上皮中的表达,分析其与胆脂瘤上皮增生调节中的可能作用。方法应用免疫组化ＳＰ染色法和计算机图像分析法,检测２２例胆脂瘤上皮和１０例正常外耳道皮肤中Ｒａｓ蛋白的表达情况。结果胆脂瘤上皮各层细胞均存在Ｒａｓ蛋白的较强表达,其中１５／２２为胞膜着色,５／２２胞膜胞浆均着色,２例仅胞浆着色,正常外耳道表皮中Ｒａｓ蛋白主要表达于胞膜,以基底层显色为主;两种组织Ｒａｓ蛋白阳性表达的平均积分吸光度分别为０．８７０和０．４６３,差异呈高度显著性。结论中耳继发性胆脂瘤上皮中存在Ｒａｓ蛋白的高表达,说明胆脂瘤上皮具有高度增生性。     

转化生长因子β1和即刻早期基因及p27在中耳胆脂瘤上皮中的表达

目的　研究转化生长因子 β1(transforminggrowthfactor β1,TGF β1)、即刻早期基因 (c fos)、周期素依赖性激酶抑制蛋白 p2 7在中耳胆脂瘤上皮中的表达及相互之间的关系 ,探讨其表达对胆脂瘤侵袭能力的影响。方法　应用免疫组化链霉菌抗生物素蛋白 过氧化酶染色法检测TGF β1、c fos和 p2 7在 31例胆脂瘤上皮、11例胆脂瘤患者外耳道上皮和 10例正常外耳道上皮中的表达 ,应用计算机图像分析系统对其阳性表达进行定量分析。结果　TGF β1和c fos在胆脂瘤上皮中表达阳性率分别为 87 1%和 83 9% ,与胆脂瘤患者外耳道上皮和正常外耳道上皮相比表达差异有显著性。而p2 7在胆脂瘤上皮和外耳道上皮组上皮中均未见阳性表达。胆脂瘤上皮中c fos与TGF β1表达无相关性 (r =0 339,P =0 331) ;胆脂瘤侵袭能力与c fos表达有高度显著相关关系 (r =- 0 96 5 ,P <0 0 1) ,与TGF β1表达也有高度相关关系 (r =- 0 4 0 6 ,P <0 0 1)。 结论　即刻早期基因c fos在胆脂瘤中表达显著增强 ,且与胆脂瘤的侵袭能力有高度显著相关性 ,提示高c fos表达是胆脂瘤高增殖特征的因素之一。TGF β1在胆脂瘤上皮中高表达亦表明其在胆脂瘤的高增殖能力方面起一定作用     

中耳胆脂瘤上皮中CYLD、c-jun、Ki-67的表达及意义

目的研究抑癌基因CYLD(cylindromatosis)、原癌基因c-jun及细胞核增殖相关抗原Ki-67在中耳胆脂瘤上皮细胞中的表达情况,分析它们之间的相互关系,探讨CYLD、c-jun、Ki-67在中耳胆脂瘤发病机制中的可能作用。方法采用免疫组化技术检测CYLD、c-jun及Ki-67蛋白产物在30例中耳胆脂瘤上皮组织(实验组)及20例正常耳后皮肤上皮组织(对照组)中的表达情况。结果 CYLD蛋白产物阳性表达定位于细胞浆,其在中耳胆脂瘤上皮中主要见基底层弱阳性或阳性表达,而在对照组正常耳后皮肤上皮全层均见阳性表达或强阳性表达,CYLD蛋白在中耳胆脂瘤上皮中的表达明显低于正常耳后皮肤(X2=16.333,p=0.000,p<0.05)。c-jun蛋白产物阳性表达定位于细胞浆和细胞核,其在中耳胆脂瘤上皮中上皮全层均见阳性表达或强阳性表达,而在对照组正常耳后皮肤上皮中均仅见基底层弱阳性或阳性表达,c-jun蛋白在中耳胆脂瘤上皮中的表达明显高于正常耳后皮肤(X2=14.901,p=0.000,p<0.05)。Ki-67蛋白产物定位于细胞核,细胞质内未见Ki-67蛋白表达。其在中耳胆脂瘤上皮基底层、棘层,甚至颗粒层中均可见阳性或强阳性表达,而在对照组正常耳后皮肤上皮中均仅见基底层弱阳性或阳性表达,Ki-67蛋白在中耳胆脂瘤上皮中的表达明显高于正常耳后皮肤(X2=13.675,P=0.000,P<0.05)。Spearman相关性分析发现中耳胆脂瘤上皮细胞CYLD、c-jun蛋白表达存在负相关(rs=-0.381,P=0.038,P<0.05)。结论与正常耳后皮肤的上皮细胞相比,中耳胆脂瘤上皮细胞具有高度增殖的能力。CYLD、c-jun异常表达可能与中耳胆脂瘤发生、发展密切相关。在中耳胆脂瘤上皮中可能也存在CYLD对c-jun表达的负调节。     

M CP -1和 FN 在中耳胆脂瘤上皮中的表达和意义

目的探讨单核细胞趋化因子（monocyte chemotactic factor ,MCP -1）和纤维连接蛋白（fibronectin , FN）在继发性中耳胆脂瘤上皮中的表达及其对胆脂瘤上皮侵袭能力的影响。方法应用免疫组织化学MaxVi-sionTM法检测MCP-1和FN在30例中耳胆脂瘤上皮、20例胆脂瘤患者耳后正常皮肤、16例非胆脂瘤患者耳后正常皮肤中的表达,应用计算机图像分析系统对其阳性表达灰度值情况及分析,比较三组之间 MCP -1和 FN表达的差异。结果 MCP-1阳性细胞表达主要分布于胆脂瘤上皮全层,其中棘层呈高表达,MCP-1在中耳胆脂瘤上皮中阳性表达率为70％,灰度值为147．2±20．1,强于胆脂瘤患者耳后正常皮肤中的阳性表达（35％,200．8±18．4）和非胆脂瘤患者耳后正常皮肤中的阳性表达（37．5％,193．3±15．5）（ P＜0．05）。FN阳性细胞主要分布于胆脂瘤上皮全层,基底层、棘层和基质呈高表达,FN在中耳胆脂瘤上皮中阳性表达率为76．7％,灰度值为139．2±18．5,强于胆脂瘤患者耳后正常皮肤中的阳性表达（30％,195．0±12．9）和非胆脂瘤患者耳后正常皮肤中的阳性表达（31．3％,191．6±13．5）（P＜0．05）。在30例中耳胆脂瘤上皮组织中,MCP-1和FN的灰度值与胆脂瘤的侵袭能力负相关（rmcp-1＝-0．682,rfn ＝-0．531,P＜0．01）,MCP-1和FN蛋白的表达不存在线性相关。结论 MCP-1和FN均在成人中耳胆脂瘤中高表达,且与中耳胆脂瘤的侵袭能力呈负相关。     

ki-67、细胞周期蛋白依赖性激酶4在胆脂瘤型中耳乳突炎中的表达及意义

目的探讨ki-67、细胞周期蛋白依赖性激酶4(CDK4)在中耳继发胆脂瘤上皮中的表达,研究它们在胆脂瘤型中耳乳突炎中的作用.方法利用免疫组化的方法,检察32例胆脂瘤上皮和11例正常外耳道皮肤中的ki-67、CDK4蛋白的表达情况.结果ki-67在胆脂瘤上皮和外耳道皮肤中均为阳性表达,阳性细胞率分别为33.2%±13.2%、14.5%±4.9%,两组之间的差异具有显著性(P＜0.05);CDK4在胆脂瘤上皮和外耳道皮肤中阳性细胞率分别为35.6%±14.6%、13.6%±5.1%,两组之间的差异具有显著性(P=0.001).结论ki-67和CDK4在胆脂瘤上皮表达增高.     

胆脂瘤上皮过度增殖行为的免疫组化研究

目的 探讨中耳胆脂瘤上皮的过度增殖性和生长方式。方法 用免疫组化SP法观察了表皮生长因子受体（epidermal growth factor receptor,EGFR），增殖细胞核抗原Ki67、Ⅳ型胶原蛋白（collagenⅣ）和层粘连蛋白（laminin,LN）在18例胆脂瘤标本的表达，并与8例外耳道正常皮肤相比较。结果 EGFR有两种染色图案，棕黄色的线状或颗粒状细胞膜强染色和颗粒状的胞浆染色，胆脂瘤标本染色均强于皮肤。在Ki67的免疫染色中，阳性细胞核为棕黄色，胆脂瘤上皮的阳性细胞核比皮肤多。CollagenIV和LN图案极其相似，为连续的宗黄色线状和（或）带状图案。4例胆脂瘤上皮基膜下结膀组织中的血管比皮肤丰富。结论 EGFR和Ki67在胆脂瘤上皮的表达从不同角度反映了胆脂瘤上皮的过度增殖性。CollagenIV和LN的表达说明胆脂瘤属良性病变，血液供应可能比皮肤丰富。     

酪氨酸蛋白激酶在胆脂瘤形成中的作用

目的探讨表皮生长因子受体(epidermicgrowthfactorreceptor,EGFR)和磷酸化酪氨酸在后天性中耳胆脂瘤的表达情况,分析酪氨酸蛋白激酶途径在胆脂瘤上皮增殖演变过程中的作用。方法应用免疫组化SP染色方法和计算机图像分析系统,连续观察10例具有典型鼓膜松弛部穿孔的后天性中耳胆脂瘤患者的不同部位(胆脂瘤上皮、鼓膜穿孔部位邻近皮肤、耳道深部正常皮肤)中EGFR和磷酸化酪氨酸的表达情况。结果EGFR和磷酸化酪氨酸在耳道深部正常皮肤、鼓膜穿孔部位邻近皮肤和胆脂瘤上皮中呈一致性连续阶梯状上升,它们在胆脂瘤上皮各层细胞均存在阳性表达,以基底层和棘层为著;穿孔部位邻近皮肤则在基底层和棘层细胞阳性表达;耳道深部正常皮肤仅基底层阳性表达。两者之间存在正相关,总相关系数为0.989(P<0.01)。结论酪氨酸蛋白激酶途径在胆脂瘤上皮增殖演变过程中起重要作用。     

转化生长因子β1和即刻早期基因及p27在中耳胆脂瘤上皮中的表达

OBJECTIVES: To analyze the expression of transforming growth factor-beta1 (TGF-beta1), cyclin dependent kinase inhibitor (p27) and c-fos in human middle ear cholesteatomas and to investigate the correlation between their expression and the ability of erosion of cholesteatoma. METHODS: Immunohistochemical staining (SP method) of 31 cholesteatomas and 11 external ear canal skin samples from patients and 10 external ear canal skin samples from healthful men which were taken intraoperatively, was performed for c-fos, TGF-beta1 and p27 positivity. The signals representing the expression of c-fos, TGF-beta1 and p27 were observed under microscope and scanned into a computer by an image scanner. The gray-scale of positive signals were quantitated by image processing computer. RESULTS: The percentage of positive expression of TGF-beta1 and c-fos in cholesteatoma were 87.1% and 83.9%, respectively. Their expression tended to be increased greatly compared with which in the skins of the control groups. Positive p27 signals were not observed in cholesteatomas and external ear skin tissues. It showed statistically significant correlation between expression of c-fos and the ability of erosion of cholesteatoma. There was correlation between the express ion of TGF-beta1 and the ability of erosion of cholesteatoma too. But there was no correlation between the expression of c-fos and TGF-beta1. CONCLUSION: The expression of c-fos in cholesteatoma was significally higher compared with which in the skin of external auditory of cholesteatoma patients and healthful men, which indicate that c-fos plays an important role in the hyperproliferative of cholesteatoma. The expression of TGF-beta1 was significant higher in cholesteatoma, which indicate that cytokines such as TGF-beta1 play a great role in the etiology of cholesteatoma.     

Nucleoplasm staining patterns and cell cycle-associated expression of Ki-67 in middle ear cholesteatoma

OBJECTIVES: To compare Ki-67 expression patterns in middle ear cholesteatoma with the corresponding retroauricular and external auditory canal skins, and to determine the cell cycle-dependent localization of Ki-67. MATERIAL AND METHODS: MIB-1 monoclonal antibody was used for comparative assessment of proliferative activity of middle ear cholesteatoma, external auditory canal skin, and retroauricular skin samples on formalin-fixed paraffin-embedded tissue sections. Primary keratinocytes from cholesteatoma tissue were isolated and subjected to kinetic analysis of the cell cycle. RESULTS: Higher proliferative activity was established in cholesteatoma in comparison with retroauricular and external auditory canal skins. Three different staining patterns have been described. Kinetic analysis revealed continuous expression of Ki-67 during all active phases of the cell cycle and remained "silent" in resting cells. CONCLUSION: The established correlation between the staining patterns and cell cycle-associated expression of Ki-67 specifies Ki-67 as a reliable and stable marker of proliferation for middle ear cholesteatoma.     

Markers of epidermal proliferation in middle ear cholesteatoma

Middle ear cholesteatoma is characterized by the presence in the middle ear cavity of a stratified squamous epithelium with keratin deposits which by constant proliferation leads to extensive bone destruction. The goal of this study was to evaluate, by immunohistochemical study, the expression of epithelial markers of proliferation--Ki-67 and PCNA in the matrix of cholesteatoma. The materials used in this study were 16 acquired cholesteatoma tissues collected from patients in the age 6-17 years during surgery. The specimens from the skin of the external ear canal were employed as the control. In the immunohistochemical specimens staining intensity and distribution of Ki67 and PCNA positive cells in various layers of the epithelium were assessed in three stages scale. The results were compared to the clinical parameters such as--type of cholesteatoma (pars flaccida or tensa), presence of ear discharge, degree of ossicular destruction and involvement of attic and mastoid. In the cholesteatoma matrix Ki-67 and PCNA positive cells were present in basal and suprabasal cell layers and also more superior layers, unlike the control skin were only basal cells show positive staining. The number of positive cells and intensity of staining was also greater in the cholesteatoma matrix than in skin of external auditory meatus. No correlation was found between results of immunohistochemical examination and clinical parameters.     

CDK4、P16在中耳胆脂瘤中的表达及意义

目的：观察细胞周期蛋白依赖性激酶CDK4、细胞周期蛋白依赖性激酶抑制因子P16在中耳胆脂瘤上皮的表达情况,探讨它们在胆脂瘤发病机制中的作用。方法：采用免疫组织化学技术SABC法检测CDK4、P16在中耳胆脂瘤35例、正常外耳道皮肤20例中的表达,并结合胆脂瘤对听小骨骨质破坏程度,采用SPSS11.5软件进行统计学分析。结果：CDK4、P16均表达于胆脂瘤上皮的棘细胞层、颗粒层和角质层,阳性率分别为68.6%、88.6%,高于对照组(前者P=0.011,后者P=0.02)。二者在胆脂瘤上皮的表达有一定的相关性,P＜0.05;与中耳胆脂瘤听小骨骨质破坏程度之间无明显相关性,P＞0.05。结论：CDK4与P16在中耳胆脂瘤发生中是共同而非单独起作用的,从细胞周期的角度可反映胆脂瘤上皮细胞过度增殖的同时也增强负调控因子来抑制增殖,使凋亡同时加快而达到新的平衡。但与骨质破坏程度可能无直接关系。     

肿瘤坏死因子-α在中耳胆脂瘤的表达及其对邻近骨质的作用

目的 :探讨肿瘤坏死因子 - α(TNF- α)在中耳胆脂瘤中的表达及其对邻近骨质的作用。方法 :应用TNF- α单克隆抗体对 18例中耳胆脂瘤组织和 8例正常外耳道、面部皮肤和鼓膜进行免疫定位检测。结果 :TNF-α定位于胆脂瘤组织的上皮及上皮下结缔组织 ,较正常外耳道及鼓膜的染色明显增强。结论 :TNF- α在中耳胆脂瘤组织中有较高表达并通过两条途径引起骨质吸收 :1TNF- α作为自分泌调节因子引起破骨性骨吸收 ;2 TNF-α作为中间信使 ,激活炎性细胞释放一系列生物酶引起骨组织脱钙 ,骨基质、骨蛋白溶解 ,最终导致骨质吸收。     

Expression patterns of p27Kip1 and Ki-67 in cholesteatoma epithelium

OBJECTIVES: The cell cycle must be involved in cell proliferation of the epithelium of middle ear cholesteatoma Cyclins and cyclin-dependent kinase (CDK) complexes have important regulatory roles during cell cycle progression. Cyclin-CDK complexes are in turn regulated by the cyclin-dependent kinase inhibitors (CDKIs), which generally inhibit cell cycle progression. One of the important CDKI members is p27(Kip1). The goal of this study is to evaluate the expression of p27(Kip1) and Ki-67, a proliferation marker, in cholesteatoma and in the skin of the external ear canal. METHODS: The expressions of p27(Kip1) and Ki-67 in cholesteatoma epithelium (n = 20) and ear canal epithelium (n = 7) were investigated by an immunohistochemical technique. RESULTS: In cholesteatoma epithelium specimens, the expression of p27(Kip1) was observed from the parabasal layer to the granular layer, but not in the basal layer. Ki-67 was expressed dominantly in the basal and parabasal cell layers. Their expressions tend to be increased compared with their expressions in the normal ear canal skin. The expression pattern of the proliferation marker Ki-67 in the epithelial layers of two groups was inversely related to the expression of p27(Kip1). CONCLUSIONS: In cholesteatoma, the expressions of CDKI and Ki-67 were both increased in this study. The ability to inhibit proliferative activity was also increased in the cholesteatoma epithelium. The expression pattern of the proliferation marker Ki-67 in the epithelial layers was inversely related to the expression of p27(Kip1). Not only is the proliferation activity increased, but also the ability to inhibit hyperproliferation is increased in the cholesteatoma epidermis. Despite increased proliferative activity in the cholesteatoma epidermis, epithelial cells still retain the capability to prevent cell cycle arrest by means of p27(Kip1).     

上皮细胞增殖和凋亡在中耳胆脂瘤发病学的意义(附33例报告)

目的 :通过探讨胆脂瘤上皮 (CE)、胆脂瘤患者外耳道上皮 (CAMS)和正常健康者外耳道上皮(NAMS)的增殖和凋亡及 p5 3基因与上皮细胞增生和凋亡的关系 ,阐明增殖和凋亡异常在胆脂瘤发病中的作用。方法 :采用免疫组织化学和原位末端标记技术检测细胞增殖标记物 PCNA、Ki- 6 7和 p5 3蛋白在 33例 CE、2 5例CAMS和 10例 NAMS细胞的表达及细胞凋亡。结果 :在上述标本中均有 PCNA表达及凋亡细胞存在 ,但不同类型上皮阳性细胞数量、染色强度及分布不同。 CE存在过度增殖和凋亡 ,CAMS亦存在过度增殖。 p5 3表达与PCNA呈正相关 ,与细胞凋亡呈负相关。结论 :CE具有过度增殖和凋亡的特性 ,增殖与凋亡的紊乱与胆脂瘤形成密切相关。     

RANKL和IL-17A在中耳胆脂瘤中的表达及相关性研究

目的观察破骨细胞分化因子（RANKL）和白介素-17A（IL-17A）在中耳胆脂瘤组织中的表达,探讨二者在中耳胆脂瘤骨质破坏机制中的作用。方法采用免疫组织化学两步法和计算机图像定量分析法,检测RAN-KL、IL-17A在25例中耳胆脂瘤上皮、16例胆脂瘤周围肉芽组织及15例正常外耳道皮肤中的表达,同时观察二者的表达与临床听小骨骨质破坏程度的相互关系。结果（1）RANKL、IL-17A在胆脂瘤上皮及胆脂瘤周围肉芽组织中表达增高,分别与正常对照组比较,差异均有统计学意义（tRANKL=9.864、5.630,均P〈0.05;tIL-17A=13.905、9.011,均P〈0.05）,且二者的表达与骨质破坏程度相关。（2）在胆脂瘤上皮及胆脂瘤周围肉芽组织中,RANKL与IL-17A的表达呈正相关（r上皮=0.692,r肉芽=0.538,P〈0.05）。结论RANKL及IL-17A在中耳胆脂瘤组织中呈高表达,与骨质破坏程度成正比,表明其与胆脂瘤骨质破坏机制密切相关;RANKL与IL-17A表达有相关性,提示二者之间可能存在相互作用。     

缺氧诱导因子-1α在中耳胆脂瘤组织中的表达

目的:探讨缺氧诱导因子- 1α(HIF- 1α)在中耳胆脂瘤发病机制中的作用,检测其在中耳胆脂瘤和外 耳道正常上皮中的表达情况。方法:取31例中耳胆脂瘤和10例外耳道正常上皮标本,用免疫组织化学技术检测 HIF-1α蛋白的表达。结果:在中耳胆脂瘤中HIF-1α的表达较外耳道正常上皮明显增多(P<0.05)。结论:中耳 胆脂瘤中HIF-1α的表达显著升高,推测HIF-1在中耳胆脂瘤的发生、发展过程中具有极其重要的作用,并且缺 氧有可能是中耳胆脂瘤发病机制中的一个诱因。