Cyclin D1在中耳胆脂瘤中的表达及意义

目的 检测细胞周期蛋白D1(Cyclin D1)在中耳胆脂瘤中的表达,探讨其在胆脂瘤发病机制中的作用.方法 采用免疫组织化学技术PV-6000二步法检测CyclinD1在30例中耳胆脂瘤组织及15例正常耳后皮肤中的表达.结果 CyclinD1在中耳胆脂瘤组织及正常耳后皮肤中的阳性率分别为70.0％、26.7％,两者比较差异有统计学意义(x2 =5.951,P＜0.05).结论 CyclinD1在中耳胆脂瘤上皮中存在高表达,这反映了胆脂瘤上皮的过度增殖性,说明CyclinD1可能在中耳胆脂瘤的发生、发展中起重要作用.     

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。     

Caspase-8在中耳胆脂瘤上皮中的表达及意义

目的 研究caspase-8(胱天蛋白酶-8)在中耳胆脂瘤上皮的表达,探讨其在中耳胆脂瘤发病机制中的作用.方法 分别采用免疫组化法和Western Blot(蛋白质印迹法)技术检测caspase-8在36例中耳胆脂瘤组织及20例正常外耳道皮肤组织中的表达情况.结果 caspase-8在36例胆脂瘤上皮各层细胞中均有强表达,而正常外耳道皮肤表达较弱,caspase-8在胆脂瘤上皮中的蛋白表达水平较正常外耳道皮肤显著增高,差异具有统计学意义(P<0.01).结论 胆脂瘤上皮中caspase-8表达较外耳道皮肤显著增高,其可能参与了中耳胆脂瘤上皮细胞的过度凋亡及增殖的调控.     

基质金属蛋白酶2和9在不同部位的中耳胆脂瘤的表达

目的 探讨基质金属蛋白酶(matrix metalloproteinase,MMP)2和9在不同部位的中耳胆脂瘤的表达差异及其骨破坏作用.方法 采用免疫组化方法 检测上鼓室胆脂瘤18例、鼓室窦胆脂瘤16例、胆脂瘤患者外耳道皮肤10例、正常外耳道皮肤10例中MMP-2和MMP-9的表达.结果 MMP-2、MMP-9在上鼓室胆脂瘤和鼓室窦胆脂瘤上皮各层细胞中均有表达,基底层表达最强;鼓室窦胆脂瘤较上鼓室胆脂瘤染色深.正常外耳道皮肤和胆脂瘤患者外耳道皮肤有非常浅的表达,主要分布在基底层细胞的胞质中.MMP-2、MMP-9在正常外耳道皮肤和胆脂瘤患者外耳道皮肤的表达差异无统计学意义(P值均<0.05);上鼓室胆脂瘤和鼓室窦胆脂瘤中MMP-2、MMP-9的表达均高于正常外耳道皮肤和胆脂瘤患者外耳道皮肤,差异有统计学意义(P值均<0.05);鼓室窦胆脂瘤中MMP-2、MMP-9的表达高于上鼓室胆脂瘤,差异具有统计学意义(P值均>0.05).胆脂瘤上皮中MMP-2和MMP-9的表达呈显著正相关(r=0.974,P<0.01),MMP-2的表达高于MMP-9.MMP-2和MMP-9的表达与听小骨的破坏程度呈正相关(r=0.789及0.803,P值均<0.01).结论 胆脂瘤型中耳炎骨质破坏过程中MMP起重要作用.鼓室窦胆脂瘤的骨质破坏程度较上鼓室胆脂瘤重,更具侵袭性,更容易产生颅内外并发症.     

中耳胆脂瘤上皮PTEN基因的表达及意义

目的探讨PTEN基因在中耳胆脂瘤形成机制中的作用。方法应用免疫组化染色和逆转录聚合酶链反应(RT-PCR)方法检测32例中耳胆脂瘤组织和10例正常外耳道皮肤组织中PTEN的表达情况。结果中耳胆脂瘤组织和正常外耳道皮肤组织均有PTENmRNA和蛋白的表达,且两组间无统计学差异(P>0.05)。结论在胆脂瘤形成过程中,PTEN的作用更趋向于维持上皮细胞的正常生理状态,而不是发生基因异常导致细胞非正常增殖。     

Toll样受体在慢性化脓性中耳炎和中耳胆脂瘤中的差异表达及其意义

目的 检测Toll样受体2( toll-like receptor 2,TLR2)和Toll样受体4(toll-like receptor 4,TLR4)在慢性化脓性中耳炎、中耳胆脂瘤中的表达,探讨其在慢性化脓性中耳炎和中耳胆脂瘤发病中的作用.方法 选取耳硬化症患者(对照组)、慢性化脓性中耳炎患者(化脓性中耳炎组)、中耳胆脂瘤患者(中耳胆脂瘤组)各30例,应用实时定量聚合酶链反应( real-time PCR)、蛋白质印迹法( Western blot)、免疫组化检测TL R2/TLR4在正常外耳道皮肤,慢性化脓性中耳炎黏膜、肉芽组织,中耳胆脂瘤患者黏膜、肉芽组织及胆脂瘤囊壁中的表达,并比较表达程度的差异.结果 ①TLR2/TLR4mRNA及蛋白质在正常外耳道皮肤,慢性化脓性中耳炎中耳黏膜、肉芽组织,胆脂瘤的中耳黏膜、肉芽组织及胆脂瘤囊壁均有表达.②TLR2/TLR4 mRNA及其蛋白质在慢性化脓性中耳炎及胆脂瘤黏膜中的表达均高于正常外耳道皮肤(P值均＜0.05),在胆脂瘤囊壁中的表达低于正常外耳道皮肤(P值均＜0.05),两组黏膜中TLR2/TLR4的表达差异无统计学意义(P值均＞0.05).③TLR2/TLR4mRNA及蛋白质在两组中耳炎肉芽组织中的表达均高于正常外耳道皮肤(P值均＜0.01),且TLR2mRNA在中耳胆脂瘤肉芽组织中的表达高于慢性化脓性中耳炎(P＜0.05).④TLR2/TLR4阳性细胞主要分布在肉芽组织中,且明显多于正常外耳道皮肤,但胆脂瘤囊壁中的阳性细胞少于正常外耳道皮肤.结论 TLR2和TLR4在正常外耳道皮肤、慢性化脓中耳炎及中耳胆脂瘤中均有表达,提示中耳具有TLR2、TLR4参与调节的固有免疫系统,但二者的差异性表达也提示其在慢性化脓性中耳炎和中耳胆脂瘤的发病中所起的作用不同.     

金属硫蛋白2及白细胞介素1α和6在中耳胆脂瘤组织中的表达及意义

目的 观察金属硫蛋白2( metallothionein 2,MT-2)及白细胞介素1α(IL-1α)、白细胞介素6(IL-6)在中耳胆脂瘤组织中的表达及相关性,探讨其在中耳胆脂瘤发生发展过程中的作用.方法 采用免疫组化和反转录聚合酶链反应(RT-PCR)技术检测25例中耳胆脂瘤组织和7例正常外耳道皮肤组织中MT-2及IL-1α、IL-6蛋白和MT-2 mRNA的表达情况并分析其相关性.进一步以胆脂瘤鳞屑刺激人角质细胞系HaCaT,观察细胞MT-2 mRNA和蛋白质的表达变化.结果 MT-2、IL-1α及IL-6在中耳胆脂瘤中的表达明显高于正常外耳道皮肤,差异具有统计学意义(P值均＜0.05);MT-2mRNA在中耳胆脂瘤和正常外耳道皮肤中的表达差异具有统计学意义(t=15.38,P＜0.05).中耳胆脂瘤组织中MT-2与IL-1α的表达呈正相关关系(r=0.856,P＜0.05),与IL-6的表达亦呈正相关关系(r=0.714,P＜0.05).在胆脂瘤鳞屑刺激下,HaCaT细胞MT-2的mRNA和蛋白质表达均有明显增强,其表达变化与鳞屑组织呈浓度依赖关系.结论 MT-2和IL-1α、IL-6的异常表达可能在中耳胆脂瘤的发生、发展过程中起一定的作用.胆脂瘤鳞屑组织可能通过促进MT-2的过量表达而参与了胆脂瘤上皮的过度增殖.     

肿瘤坏死因子受体1在中耳胆脂瘤上皮中的表达

目的 研究肿瘤坏死因子受体1（TNFR1）在中耳继发性胆脂瘤上皮的表达，探讨其在胆脂瘤型中耳炎的发病机制中的作用。方法 分别采用免疫组化法和Western blot技术检测TNFR1在36例中耳胆脂瘤组织及20例正常外耳道皮肤组织中的表达情况。结果 TNFR1在36例胆脂瘤上皮各层细胞中均有强表达，定位于胞膜和胞质，而外耳道皮肤表达较弱甚至没有表达，TNFR1在胆脂瘤上皮中的蛋白表达水平较外耳道皮肤显著增高，差异有统计学意义（P〈0．01）。结论 胆脂瘤上皮中TNFR1表达较外耳道皮肤显著增高，肿瘤坏死因子可能通过与TNFR1相结合而导致胆脂瘤上皮细胞的过度增殖及凋亡。     

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。     

MMP-9在中耳胆脂瘤中的表达和意义

目的 研究基质金属蛋白酶-9(matrix metalloproteinase,MMP-9)在中耳胆脂瘤上皮中的表达,并探讨其与中耳胆脂瘤的血管形成以及胆脂瘤侵蚀行为的关系.方法采用免疫组织化学SP法检测MMP-9、Ⅳ型胶原蛋白在44例患者中耳胆脂瘤组织标本与10例正常外耳道组织中的表达.结果 MMP-9在胆脂瘤上皮各层均有表达,在外耳道皮肤中为弱阳性表达;胆脂瘤基质周围微血管计数均数高于正常外耳道皮肤,二者差异具有统计学意义(P＜0.05);MMP-9在胆脂瘤中的表达与周围微血管计数间呈正相关关系.结论 MMP-9可能在中耳胆脂瘤血管生成中起重要作用.     

Activation of the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway in human cholesteatoma epithelium

Cholesteatoma is a benign keratinizing squamous epithelial lesion characterized by the hyper-proliferation of keratinocytes with abundant production of keratin debris in the middle ear. The epidermal growth factor receptor (EGFR)/Akt/nuclear factor-kappa B (NF-κB)/cyclinD1 signaling pathway is one of the most important pathways in regulating cell survival and proliferation. We hypothesized that the EGFR/Akt/NF-κB/cyclinD1 signaling pathway may be activated and involved in the cellular hyperplasia mechanism in acquired cholesteatoma epithelium. Immunohistochemical staining of phosphorylated EGFR (p-EGFR), phosphorylated Akt (p-Akt), activated NF-κB and cyclinD1 protein was performed in 40 cholesteatoma samples and 20 samples of normal external auditory canal (EAC) epithelium. Protein expression of p-EGFR, p-Akt, activated NF-κB and cyclinD1 in cholesteatoma epithelium was significantly increased when compared with normal EAC epithelium (p < 0.01). In cholesteatoma epithelium, a significant positive association was observed between p-EGFR and p-Akt expression and between the expressions of p-Akt and NF-κB, NF-κB and cyclinD1, respectively (p < 0.01). No significant relationships were observed between the levels of investigated proteins and the degree of bone destruction (p > 0.05). The increased protein expression of p-EGFR, p-Akt, NF-κB and cyclinD1 and their associations in cholesteatoma epithelium suggest that the EGFR/Akt/NF-κB/cyclinD1 survival signaling pathway is active and may be involved in the regulatory mechanisms of cellular hyperplasia in cholesteatoma epithelium.     

Comparative analysis of the expression of E-cadherin, β-catenin,and β1 integrin in congenital and acquired cholesteatoma

E-cadherin, β-catenin, and β1 integrin are important cell adhesion molecules to maintain epithelial structure and function. We investigated the expression of these cell adhesion molecules in cholesteatomas to understand the role of cell–cell and cell–extracellular matrix interaction in cholesteatomas. An immunohistochemical investigation was carried out on 35 cholesteatoma tissue samples (14 congenital, 21 acquired cholesteatomas) and 10 normal retroauricular skin (RAS) tissues which are obtained during middle ear surgery. The expression rate was measured to find out differences between retroauricular skin and cholesteatoma, as well as between congenital and acquired cholesteatoma. E-cadherin expression rate was significantly lower in the cholesteatoma (spinous layer 88.7 ± 17.9 %, granular layer 54.6 ± 22.6 %) than in the RAS (100 %, 74.4 ± 7.4 %) and in the acquired (83.3 ± 19.4 %, 48.1 ± 22.9 %) than in the congenital (96.7 ± 12.0 %, 64.4 ± 18.8 %). β-catenin expression rate was significantly lower in the cholesteatoma (spinous layer 84.1 ± 17.2 %, granular layer 28.7 ± 30.8 %) than in the RAS (100 %, 75.9 ± 6.1 %) and in the acquired (78.1 ± 17.0 %, 17.1 ± 22.3 %) than in the congenital (93.2 ± 13.5 %, 46.1 ± 34.2 %). The expression pattern of β-catenin is similar to that of E-cadherin. In β1 integrin, there was no significant difference of the expression rate between RAS and cholesteatoma, as well as between congenital and acquired cholesteatoma. In conclusion, the expression of E-cadherin and β-catenin is reduced in cholesteatoma, and the reduction is more pronounced in acquired cholesteatoma than in congenital cholesteatoma. Acquired cholesteatomas showed more aggressive characteristics than congenital cholesteatomas in terms of cell–cell adhesion.     

Molecular biological diagnosis of congenital and acquired cholesteatoma on the basis of differences in telomere length

OBJECTIVE: To establish a molecular biological basis for differentiation of congenital and acquired cholesteatoma. STUDY DESIGN: The time of onset was estimated for congenital cholesteatoma and for acquired cholesteatoma by comparing the telomere length and the telomerase activity in the tissues of both diseases with the values of those parameters in normal external ear canal skin. METHODS: The telomere length was determined by extracting DNA from each tissue and then applying the Southern blot technique to hybridize it with a 32P-labeled telomeric oligonucleotide (TAAGGG)8 probe. The telomerase activity was analyzed by a modification of the polymerase chain reaction-based telomeric repeat amplification protocol. RESULTS: The telomere length in congenital cholesteatoma tissue was shorter than the length in normal external ear canal skin from the same patient, whereas in acquired cholesteatoma tissue the telomere length was almost the same as in the normal external ear canal skin. Some of the acquired cholesteatoma tissue specimens and normal external ear canal skin specimens were positive for telomerase activity, but all of the specimens of congenital cholesteatoma tissue were negative for telomerase activity. No correlation was found between the presence of telomerase activity and the telomere length. CONCLUSIONS: The present results indicate that congenital cholesteatoma manifests at an earlier time compared with acquired cholesteatoma, and the results can be thought to support the theory that congenital cholesteatoma originates from vestigial fetal tissue or aberrant tissue. In addition, the finding that telomerase activity was weak in the congenital cholesteatoma tissue suggests the possibility that vestigial fetal tissues and aberrant tissues are naturally eliminated in normal subjects as a result of apoptosis.     

Cell cycle inhibitory protein p27 in human middle ear cholesteatoma

AIM: To evaluate the immunohistochemical and molecular presentation of protein p27 in cholesteatoma. METHODS: 42 cholesteatoma samples and 6 external ear canal skin (EECS) specimens were investigated and analyzed taking into consideration congenital, acquired, recurrent cholesteatoma, and EECS. RESULTS: The expression of p27 was found in 16 (38.1%) out of 42 specimens of cholesteatoma and in 5 (83.3%) out of 6 specimens of EECS. There was a significant difference in p27-positive staining rate between EECS and cholesteatoma epithelium (p < 0.008). The presence of p27 was detected in 10 cases of acquired cholesteatoma, 2 cases of congenital and 3 cases of recurrent cholesteatoma. There was no significant difference between the presence of p27 in cholesteatoma and EECS (p = 0.01). CONCLUSION: The down-regulation of p27 is a key player in cell cycle control and plays an undefined role in the pathogenesis of all types of cholesteatoma.     

中耳胆脂瘤中P-Akt和Foxo3的表达及其和凋亡的关系

目的 检测人类叉头框O3(forkhead box O3,Foxo3)蛋白、磷酸化蛋白激酶B(phosphorylated protein ki-nase B,P-Akt)在中耳胆脂瘤的表达和DAPI核染色情况.方法 应用免疫荧光技术(immunofluorescence,IF)检测中国医科大学附属盛京医院2019年8...     

Argyrophilic nucleolar organizer regions in auditory meatal skin and middle ear cholesteatoma

Comparative silver-staining of argyrophilic nuclear organizer regions (AgNORs) was performed to study the proliferative activity of auditory meatal skin and middle ear cholesteatoma. AgNOR expression patterns were counted by standardized methods in two centres, Bochum and London, and mean numbers of dots per nucleus were calculated. Specimens of normal auditory meatal skin showed 1.54 dots/nucleus (n = 12) in the Bochum study, whereas cholesteatoma had 3.71 dots/nucleus (n = 21). In the London study normal meatal skin showed two dots/nucleus (n = 4), whereas acquired cholesteatoma (n = 8) gave a mean of 4.90 dots/nucleus and congenital cholesteatoma a mean of 4.70 dots/nucleus (n = 2). Our findings confirm the hyperproliferative state of middle ear cholesteatoma, suggest that the congenital variety of cholesteatoma may have a similar activity and indicate that AgNOR staining is a useful technique for assessing cellular proliferation in cholesteatoma and objectifying and quantifying its aggressive behaviour.     

PTEN及P-ERK和P-AKT在中耳胆脂瘤上皮中的表达及意义

目的：检测蛋白酪氨酸磷酸酶基因（PTEN）、磷酸化蛋白激酶B（P-AKT）和磷酸化细胞外信号调节激酶（P-ERK）在人类中耳胆脂瘤上皮的表边情况及其相关性,探讨它们在中耳胆脂瘤形成机制中的重要作用。方法：应用免疫组织化学SABC法检测40例中耳胆脂瘤标本及15例正常皮肤标本中PTEN、P-AKT和P-ERK蛋白的表达量及定位。应用Western blot免疫印迹法检测其中20例中耳胆脂瘤标本和10例正常皮肤标本PTEN、P-AKT和P-ERK蛋白以及内参GAPDH的表达量。结果：①免疫组织化学显示,PTEN在胆脂瘤和正常皮肤中的细胞核及胞质均有着色。核PTEN在胆脂瘤的阳性表达率明显低于正常皮肤,两者差异具有统计学意义（P〈O．01）;质PTEN在胆脂瘤的阳性表达率明显低于正常皮肤,两者差异具有统计学意义（P〈0．01）;P-AKT主要在细胞质着色,胆脂瘤中的阳性表达率明显高于正常皮肤,两者差异具有统计学意义（P〈0．01）;p-ERK主要在细胞核着色,胆脂瘤中的阳性表达率明显高于正常皮肤,两者差异亦具有统计学意义（P〈O．01）。在中耳胆脂瘤标本中,PTEN分别与P-AKT、P-ERK蛋白的表达之间呈显著负相关（P〈O．01）。②Western blot免疫印迹法检测显示：胆脂瘤中PTEN的表达量明显少于正常皮肤中的表达量;而P-AKT和P-ERK在胆脂瘤中的表达量明显多于它们在正常皮肤中的表达量。结论：PTEN、P-AKT和P-ERK蛋白在中耳胆脂瘤中的异常表达可能与胆脂瘤上皮的高度增殖和抗凋亡密切相关。PTEN表达缺失导致其抑制作用减弱,一方面使P-AKT表达过度,继而引起胆脂瘤上皮细胞凋亡受抑制;同时也使P-ERK表达过度,导致胆脂瘤上皮细胞增殖加强。     

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。