Expression of PTHrP and RANKL in acquired middle ear cholesteatoma epithelium

Abstract

Background: Regarded as the most important clinical characteristic of middle ear cholesteatoma, the exact mechanism of bone resorption in cholesteatoma still remains unknown.

Objectives: To investigate protein expression of PTHrP and RANKL in acquired middle ear cholesteatoma epithelium and analyze their functional roles in the etiopathogenesis of bone resorption in middle ear cholesteatoma.

Material and methods: A total of 22 patients who underwent surgical treatment for middle ear cholesteatoma were recruited in the study. Protein expression of PTHrP and RANKL in middle ear cholesteatoma and normal postauricular skin was investigated by immunohistochemical staining. Correlations between bone resorption degree and expression of PTHrP and RANKL were also analyzed.

Results: Protein expression of PTHrP and RANKL in cholesteatoma epithelium significantly increased when compared with normal postauricular skin epithelium. In cholesteatoma epithelium, a significantly positive association was observed between PTHrP and RANKL expression. Meanwhile, obviously positive correlations between protein expression of PTHrP and RANKL and bone resorption degree were discovered.

Conclusions and significance: The increased protein expression of PTHrP and RANKL in cholesteatoma epithelium, and their associations with the degree of bone resorption, revealing that PTHrP might promote bone resorption process in middle ear cholesteatoma through RANKL signaling pathway.     

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。     

破骨细胞分化因子及其相关因子在中耳胆脂瘤组织的表达

目的探讨破骨细胞分化因子（nuclear factor-kappa B ligand,RANKL）和其配体核因子-κB受体活化因子（receptor activator of nuclear factor-kappa B,RANK）及假性受体骨保护素（osteoprotegerin,OPG）即RANK-RANKL-OPG系统在中耳胆脂瘤的作用,儿童中耳胆脂瘤较成人骨质破坏严重的原因。方法应用免疫组织化学SP法观察RANKL和OPG在21例成人中耳胆脂瘤组织、17例儿童中耳胆脂瘤组织和18例正常外耳道深部皮肤中的表达,对染色结果进行量化处理和统计学分析。结果RANKL和RANKL/OPG比值在胆脂瘤的表达量明显高于外耳道皮肤,儿童胆脂瘤高于成人,差异有统计学意义（P〈0.05）。结论①RANKL和RANKL/OPG比值的增高与胆脂瘤的骨质破坏有关,在中耳胆脂瘤骨质破坏的机制中起重要作用;②儿童中耳胆脂瘤RANKL/OPG比值较高,这可能是导致儿童中耳胆脂瘤比成人发展快,骨质破坏严重的一个重要原因。     

慢性化脓性胆脂瘤型中耳炎的高分辨CT诊断

目的探讨高分辨CT(HRCT)对慢性化脓性中耳炎(胆脂瘤型)的诊断价值.方法对238例经手术及病理证实的中耳胆脂瘤的CT征象与手术所见进行对照研究.结果(1)CT诊断胆脂瘤的敏感性最高在上鼓室(81.25%),最低在乳突(50%).(2)CT显示胆脂瘤的病理改变为①鼓室乳突腔内胆脂瘤影;②胆脂瘤周低密度环;③鼓室乳突腔边缘骨质硬化;④鼓室乳突腔扩大;⑤听骨链破坏.结论CT检查对于中耳胆脂瘤的诊断及指导手术有很大帮助.     

两种基质金属蛋白酶在胆脂瘤和中耳癌中的表达

目的探讨基质金属蛋白酶（matrix metalloproteinases, MMP）的MMP-2和MMP-9在胆脂瘤和中耳癌中的表达差异及其在骨浸润作用。方法采用免疫组织化学方法检测36例胆脂瘤上皮,10例胆脂瘤外耳道皮肤、16例中耳癌组织及正常组织中MMP-2和MMP-9的表达。结果①MMP-2和MMP-9在胆脂瘤上皮染色最强。而且在上皮组织的各层细胞均有表达,基底膜染色最强。②胆脂瘤上皮中的MMP-2和MMP-9的表达明显高于其对应的外耳道皮肤和正常外耳道上皮。MMP-2阳性表达在胆脂瘤上皮中为83%,胆脂瘤外耳道上皮为50%,正常外耳道皮肤2例有微弱表达。③16例中耳鳞癌组织中,MMP-9的阳性表达率为50%（8/16）,MMP-2的阳性表达率为56.25%（9/16）,表达水平与肿瘤的病理分级和临床分期有关。④中耳癌组织中MMP-2的表达与MMP-9表达不一致,而胆脂瘤组织中MMP-2和MMP-9的表达密切相关。结论胆脂瘤向周围骨质侵袭的特性与中耳癌相似,MMPs家族及其抑制剂之间的平衡紊乱所致的蛋白水解酶活性增高是引起中耳胆脂瘤骨吸收的原因之一。     

蛋白酪氨酸磷酸酶基因和磷酸化蛋白激酶B在中耳胆脂瘤上皮中的表达和意义

目的 检测蛋白酪氨酸磷酸酶基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)、磷酸化蛋白激酶B(phosphorylated protein kinase B,P-AKT)在人类中耳胆脂瘤上皮组织中的表达情况及其相关性,探讨他们在中耳胆脂瘤上皮细胞形成机制中的重要作用.方法 应用免疫组织化学SP法检测40例人中耳胆脂瘤组织标本及15例正常皮肤标本中PTEN、P-AKT蛋白的表达.结果 免疫组化显示细胞质PTEN主要在胆脂瘤组织和正常皮肤组织的上皮细胞浆着色,阳性表达率为50.0％,明显低于正常皮肤组的86.6％ (x2=5.075,P＜0.01);P-AKT蛋白在中耳胆脂瘤上皮组织和正常皮肤组的上皮细胞浆着色,阳性表达率分别为72.5％和26.7％,两者差异具有显著性(x2=7.605,P=0.006,P＜0.01),15例外耳道正常上皮组织中,PTEN与P-AKT蛋白的表达之间呈显著负相关(r=-0.689,P＜0.0001,P＜0.01).结论 PTEN和P-AKT蛋白在中耳胆脂瘤上皮组织中的异常表达可能与胆脂瘤上皮的高度增殖和抗凋亡状态密切相关.PTEN蛋白表达缺失导致其抑制作用减弱,使P-AKT蛋白表达过度,继而引起胆脂瘤上皮细胞凋亡受抑制.     

Micronucleus frequency in acquired middle ear cholesteatoma

Objective—To determine the micronucleus (MN) frequency of acquired cholesteatoma tissue using an MN assay.

Material and Methods—Eighteen patients were diagnosed as having chronic otitis media with acquired cholesteatoma and were divided into primary and secondary acquired cholesteatoma groups. Cholesteatoma tissue and normal tissue specimens from the external ear canal skin were taken from the patients during surgical operations. MN frequencies of cholesteatoma and control samples were determined according to standard criteria.

Results—The MN frequencies of the cholesteatoma and control tissues were 0.54%±0.31% and 0.24%±0.11%, respectively (p<0.01). MN frequencies for the primary and secondary acquired cholesteatoma groups were 0.63%±0.36% and 0.46%±0.26%, respectively (p>0.05). MN frequencies in cholesteatoma patients without and with complications were 0.42%±0.19% and 0.85%±0.37%, respectively (p<0.05).

Conclusion—MN frequencies were found to be increased in cholesteatoma tissues when compared with external ear canal skin. The MN frequency in five cases with complications was higher than in cases without complications. These results indicate that there could be associations between MN frequency and acquired cholesteatoma and between MN frequency and complications.     

罕见中耳巨大胆脂瘤1例

中耳胆脂瘤是一种常见的慢性中耳疾病,特征是中耳腔、乳突腔及岩尖存在高度增殖的角化鳞状上皮和邻近骨质的破坏吸收,上皮细胞脱落、上皮碎屑堆积而形成胆脂瘤[1].现将我科收治的1例罕见巨大胆脂瘤病历资料报告如下.     

中耳胆脂瘤中核因子-κB的表达与活化

目的研究中耳胆脂瘤中核因子-κB(nuclearfactorkappaB,NF-κB)的表达与活化,深入阐明胆脂瘤的发病机制。方法中耳手术中收集21例中耳胆脂瘤组织及8例正常外耳道皮肤组织,分别采用免疫组化及凝胶电泳迁移阻滞法(electrophoreticmobilityshiftassay,EMSA)检测2种组织NF-κB的蛋白表达分布及DNA结合活性,并进一步以胆脂瘤鳞屑刺激人角质形成细胞系HaCaT,观察细胞NF-κB的活性变化。结果胆脂瘤上皮组织中NF-κB的表达强度显著高于正常表皮组织,其阳性细胞平均积分吸光度分别为0·168±0·051、0·088±0·019(t=4·211,P<0·01),部分胆脂瘤(12/21)上皮细胞出现明显NF-κB的核转位;EMSA结果显示胆脂瘤组电泳带相对密度扫描值平均为(16·5±10·1)%,正常皮肤组则为(1·38±1·24)%,提示胆脂瘤组织中NF-κB的DNA结合活性显著高于正常皮肤组织(t=3·600,P=0·014);在胆脂瘤鳞屑刺激下,HaCaT细胞出现NF-κB的活化,其活性变化与鳞屑组织呈剂量依赖关系。结论NF-κB在胆脂瘤组织中存在异常活化,核因子-κB可能在胆脂瘤的发生和持续发展中起重要作用。     

Localization of fibronectin in human middle ear cholesteatoma

Summary Fibronectin was localized in human cholesteatoma tissues by immunohistochemical methods. The avidin-biotin-peroxidase complex staining method was used with specific fibronectin antibody. Fibronectin appeared to be localized in the matrix of the cholesteatoma studied, particularly on the surface of the cell membranes and the nuclei of the basal cells and in connective tissue. Fibronectin was not seen in the granular layer or in the keratin area. Fibronectin was found on the surface of granulation tissue, mononuclear cells, fibroblasts and endothelial cells of blood vessels. These findings were confirmed by the immunofluorescent staining method. Our previous study showed that fibronectin induced a migration of keratinocytes, macrophages and fibroblasts demonstrated by the Boyden's chamber chemotaxis assay. Macrophages and fibroblasts were shown to produce collagenase, a bone resorption factor, in cholesteatomatous tissue. The present study showed the presence of fibronectin in the matrix of cholesteatoma and granulation tissue, suggesting that fibronectin might play an important role in the clinical development and invasive behavior of cholesteatoma.     

中耳乳突珍珠型胆脂瘤新生血管的观察

目的了解珍珠型中耳乳突胆脂瘤微血管的组织病理学特点,探讨微血管在胆脂瘤形成中的作用。方法对6例珍珠型中耳胆脂瘤微血管的病理学特点进行观察,并应用免疫组化法对其血管内皮细胞中Ⅷ因子相关抗原和CD341的表达进行观察。结果在胆脂瘤的囊壁及其蒂部含丰富薄壁微血管,Ⅷ因子相关抗原和CD34在血管内皮细胞内有明显表达。结论珍珠型胆脂瘤微血管的形成可能是胆脂瘤生长扩张的重要前提之一。     

Platelet-derived growth factor in middle ear cholesteatoma

Platelet-derived growth factor (PDGF) was localized in human middle ear cholesteatoma tissue by an immunoperoxidase technique using rabbit anti-human PDGF IgG. PDGF was found mainly in basal cells and in granulation tissue, and especially involved monocytes and fibroblast-like cells. The external ear canal epithelium was not significantly stained by anti-human PDGF. Findings demonstrate that the presence of PDGF in cholesteatoma is in response to inflammation and wound healing in the middle ear. PDGF in vitro was found to stimulate protein synthesis and cellular terminal differentiation of basal keratinocytes. PDGF also stimulated monocytes to form multinucleated osteoclast-like cells. These multinucleated cells, in turn, induced the resorption of devitalized bovine bone. This bone resorption was seen in co-cultures of osteoblasts and multinucleated osteoclast-like cells in the presence of PDGF, suggesting that cell-to-cell interaction plays a role in bone resorption. The present study suggests that PDGF takes part in the clinical development and the destructive effect of cholesteatoma.     

中耳先天性胆脂瘤1例

<正>中耳先天性胆脂瘤是少见疾病,常因病变隐匿而导致临床上延误诊断和治疗,随着影像学技术的提高和显微镜检查的普及,发现报道的先天性胆脂瘤病例逐渐增多。先天性胆脂瘤早期可无任何表现,逐渐出现传导性聋、耳痛,听力进行性下降,严重者可出现面瘫和神经性聋,甚至出现颅内并发症[1],由于无流脓、鼓膜穿孔等耳部症状,单侧的听力下降容易被忽略,诊断不易做出。现将解放军405医院耳鼻喉科2014年3月收治的先天性胆脂     

人乳头状瘤病毒及其相关基因在中耳胆脂瘤中的表达

目的　探讨人乳头状瘤病毒 (humanpapillomavirus ,HPV)感染在中耳胆脂瘤发生发展中的作用。方法　运用共同引物聚合酶链反应 (polymerasechainreaction ,PCR)和核酸分子斑点杂交法对44例 ( 44耳 )中耳胆脂瘤标本组织中的HPVDNA进行检测 ,并结合其中 35例 ( 35耳 )的病理学检查结果进行对比分析。结果　 12耳 ( 34.3 % )中耳胆脂瘤组织中观察到了HPV感染的损害特征 ;用共同引物PCR法及核酸分子斑点杂交法对 44耳中耳胆脂瘤组织标本进行HPVDNA扩增的阳性率分别为2 9 .5 % ( 13 44 )及 2 5 .0 % ( 11 44 ) ;表现有人乳头瘤病毒损害特征的 12耳中耳胆脂瘤组织HPVDNA检测阳性率为 5 8.3% ( 7 12 ) ,而无此损害特征的 2 3耳中耳胆脂瘤组织HPVDNA检测阳性率为 13.0 %( 3 2 3) ,统计学检验差异有显著性 ( χ2 =7.92 6 ,P <0 .0 0 5 )。结论　HPV感染可能激发中耳胆脂瘤上皮的分裂增殖 ,在中耳胆脂瘤发生发展中起一定的作用 ;侵蚀性乳头瘤样生长和空晕细胞改变可以作为中耳胆脂瘤组织中HPV感染的病理学证据     

血管内皮生长因子在中耳胆脂瘤中的表达及其临床意义

目的本研究的主要目的是分析与正常中耳黏膜和外耳道皮肤相比血管内皮生长因子(VEGF)在中耳胆脂瘤中的定位和分布.探讨VEGF与中耳胆脂瘤的血管形成以及胆脂瘤侵蚀性行为的关系.方法使用免疫组织化学的方法研究VEGF的表达;使用自动影像分析系统计算所形成血管的数目(Ⅳ型胶原蛋白染色).结果胆脂瘤上皮层基底部角质细胞可见VEGF的免疫反应活性呈强阳性.胆脂瘤基质周围与正常中耳黏膜上皮下结缔组织和正常外耳道皮肤相比,VEGF的免疫反应活性有显著性差异.随着胆脂瘤基质周围炎症程度的增加,其微血管计数的均数也相应地增加,由9.3±3.6血管数/mm2(炎症Ⅰ级)增加到21.1±3.4血管数/mm2(炎症Ⅱ级)和32.5±9.5血管数/mm2(炎症Ⅲ级)(p＜0.05).结论VEGF诱导的新生血管形成能够使角质细胞迁移进入中耳腔.因此,新生血管形成是中耳胆脂瘤侵蚀性行为的一个关键性的因素.     

中耳胆脂瘤上皮过度增殖与MDM2的相关性

目的探讨MDM2（murine double minute）在中耳胆脂瘤上皮增殖机制中的作用,了解胆脂瘤的过度增殖性和生长方式。方法应用免疫组织化学技术检测32例中耳胆脂瘤组和10例正常对照组中MDM2的表达。结果MDM2分布于胆脂瘤上皮全层,但在基底层细胞表达最弱,棘细胞层、颗粒层和角质层依次递增,染色最强的部位是颗粒层和角质层,而正常对照组未见MDM2分布。结论p53／MDM2反馈环异常可能是胆脂瘤上皮异常增殖和凋亡的原因之一。在胆脂瘤基底层细胞表达少量的MDM2可能是维持胆脂瘤不同于恶性肿瘤组织的无限增殖和浸润的基础。     

硬质耳内镜在胆脂瘤中耳炎手术治疗中的应用

目的探讨耳内镜在胆脂瘤中耳炎手术治疗中的作用。方法2003—2005年的中耳胆脂瘤患者43例43耳,应用硬性耳内镜协助下行乳突根治 Ⅰ期鼓室成形术,并在耳内镜下随访2年以上(平均2.211±0.323年)。结果术后1.5～3月干耳40例,鼓膜色泽正常。2例内陷袋形成,术后1年经耳内镜手术探查,发现为中耳粘连导致上中鼓室通气不畅。1例术后0.5年因患耳流脓再次手术,为修补之鼓膜与鼓窦口上皮粘连,后鼓室有孤立胆固醇肉芽肿,清除粘连及病变组织后2月干耳。随访测听结果:听力提高10～20dB21耳,21～30dB15耳,>30dB4耳,<10dB3耳。结论耳内镜能够弥补耳显微镜的不足,辅助清除中耳病灶以增加手术成功率。     

微血管密度在中耳胆脂瘤上皮增殖中的意义

目的　研究微血管密度在中耳继发性胆脂瘤上皮增殖演变过程中的意义。方法　应用免疫组化ABC染色方法和计算机图像分析系统,连续观察 15例具有典型鼓膜后皱襞处穿孔的中耳胆脂瘤患者的不同部位(鼓膜穿孔部位邻近皮肤和耳道深部正常皮肤 )中血管参数的改变,并和20例非胆脂瘤型中耳炎鼓膜穿孔部位邻近皮肤作对比。结果　胆脂瘤型中耳炎患者的鼓膜穿孔部位邻近皮肤的微血管计数、微血管相对面积 ( x±s)分别为 14.395±2.000和 (9.927±2.600 ) %,显著高于其自身耳道深部正常皮肤的 6 218±0 949和 ( 5.076±0.807 )% (P<0. 001 );亦显著高于非胆脂瘤型中耳炎鼓膜穿孔部位邻近皮肤的 6.163±1 051和 ( 5.785±1.428 )% (P<0.001 )。结论中耳胆脂瘤的鼓膜穿孔部位邻近皮肤增生活跃。     

细胞周期调控因子在中耳胆脂瘤上皮中的表达

目的根据细胞周期调控基因表达情况探讨中耳胆脂瘤上皮细胞增殖的分子机制。方法采用免疫组织化学方法检测胆脂瘤上皮细胞和胆脂瘤患者外耳道上皮细胞中周期蛋白依赖性激酶4(cyclindependentkinase4,CDK4)及其抑制因子p15、p16的表达,并结合炎症及骨质破坏程度作统计学分析。结果CDK4、p16在胞核、胞浆以棕黄色或棕褐色颗粒表达,并以胞核为主,而p15仅以胞核棕黄色或棕褐色颗粒表达。与皮肤相比,CDK4、p15、p16在胆脂瘤上皮细胞的表达显著增强,上皮下重度炎症可增强CDK4的表达;不同的骨质破坏程度上述指标的表达无显著性差异。结论胆脂瘤上皮细胞增殖活性增强,同时抑制细胞增殖的机制也增强;局部炎症可增强胆脂瘤上皮细胞的增殖活性。