中耳胆脂瘤上皮细胞增生与凋亡状态的研究

目的 :研究中耳胆脂瘤上皮细胞增生和凋亡的状态。方法 :应用免疫组化染色 SABC技术及原位凋亡细胞标记技术 (TU NEL 法 ) ,对 2 0例中耳胆脂瘤上皮组织和 10例外耳道正常上皮组织样本进行研究。结果 :在中耳胆脂瘤上皮中 ,增殖细胞核抗原 (PCNA )阳性细胞大量存在于基底细胞层、棘细胞层及颗粒细胞层 ,而在正常外耳道上皮中 PCNA阳性细胞仅存在于基底层 ;在胆脂瘤上皮中 PCNA阳性细胞率和平均光密度分别为(36 .91± 2 2 .77) %和 0 .2 4 2 7± 0 .0 5 86 ,明显高于正常外耳道上皮中的 PCNA阳性细胞率 (10 .2 5± 2 .6 5 ) %及平均光密度 (0 .1340± 0 .0 36 3) ,其差异有显著性意义 (P<0 .0 5 )。同时 ,在胆脂瘤上皮中 ,凋亡细胞存在于棘细胞层及颗粒细胞层 ,而外耳道正常上皮与之相似 ,但两者的凋亡率分别为 (2 7.5 0± 12 .5 0 ) %和 (9.96± 3.86 ) % ,其差异有显著性意义 (P <0 .0 5 )。结论 :中耳胆脂瘤上皮具有高度增生和凋亡的能力 ,并因上皮细胞的增生、分化、凋亡导致角化碎片累积而形成胆脂瘤     

热休克蛋白70在中耳胆脂瘤中的异常表达

目的研究热休克蛋白(HSP70)在中耳胆脂瘤组织中的异常表达,探讨HSP70与胆脂瘤的关系及其作用.方法采用免疫组化SABC染色法、计算机图像定量分析法,对25例中耳胆脂瘤和10例正常外耳道皮肤中HSP70的表达情况进行观察.结果在25例胆脂瘤上皮组织的全层及上皮下结缔组织中炎性细胞和成纤维细胞的胞浆中均有HSP70强表达,部分核中也有强表达.10例正常外耳道皮肤中HSP70为弱表达.计算机图象分析仪定量分析,结果显示胆脂瘤上皮中HSP70阳性细胞的平均光密度为0.3309±0.0070,正常外耳道上皮为0.1798±0.0020,胆脂瘤上皮中HSP70的含量显著高于外耳道皮肤(P＜0.001).结论HSP70可能参与了胆脂瘤的形成和发展.     

基质金属蛋白酶-1及白细胞介素-1α mRNA在中耳胆脂瘤上皮中的表达

目的 探讨基质金属蛋白酶(matrix metalloproteinases, MMP1)及其组织抑制剂(tissue inhibitor of metalloproteinases, TIMP1)和白细胞介素-1α mRNA(interleukin-1-alpha messenger ribonucleic acid, IL-1α mRNA)在中耳继发性胆脂瘤上皮的表达,分析其表达在胆脂瘤上皮溶骨性破坏中的作用.方法分别应用免疫组化SP染色法和原位杂交技术检测MMP1、TIMP1和IL-1α在32例胆脂瘤和14例胆脂瘤患者外耳道皮肤中的表达情况.结果 MMP1在32例胆脂瘤上皮各层细胞均存在较强表达,定位在胞膜和胞浆,外耳道皮肤表达较弱;两种组织MMP1阳性表达的平均(±s,下同)吸光度(A值)分别为(2 018.26±174.89)和(1 428.35±123.39),差异有极显著性(t=10.69,P<0.01);TIMP1在两者中未见明显表达;外耳道皮肤仅基底层细胞可见散在IL-1α mRNA阳性表达细胞,平均吸光度(A值)为(566.27±148.94),胆脂瘤上皮除基底层细胞外,部分区域基底上层细胞也见阳性表达,平均吸光度(A值)为938.91±223.46,差异有极显著性(t=5.03,P<0.01).结论①MMP1在胆脂瘤上皮中高表达,并提示有MMP1和TIMP1表达失衡,与胆脂瘤骨吸收特性有关.②胆脂瘤上皮高表达的IL-1α可能是MMPs过表达的原因.     

IL-8和IL-6在中耳胆脂瘤上皮中的表达

张宏伟陈乾美叶惠平林尚泽梁文妹【摘要】目的探讨白细胞介素-8(IL-8)、白细胞介素-6(IL-6)在中耳胆脂瘤上皮中的表达,分析它们在胆脂瘤上皮骨质破坏中的可能作用。方法应用免疫组织化学SABC法,检测31例患者胆脂瘤上皮和14例患者外耳道皮肤中的IL-8、IL-6的表达情况。结果IL-8主要在31例患者胆脂瘤基底上层细胞和基底层细胞阳性表达,8例患者外耳道皮肤有不同程度表达,两种组织表达的平均吸光度(A值)分别为155.07±13.93、202.15±14.32,有显著性差异(t=10.56,P<0.01)。IL-6主要在30例患者胆脂瘤基底上层细胞和基底层细胞表达,10例患者外耳道皮肤有不同程度表达,两种组织表达的平均吸光度(A值)分别为162.56±20.05、204.15±18.38,有显著性差异(t=6.63,P<0.01)。IL-8与IL-6表达间存在正相关。结论IL-8、IL-6在胆脂瘤上皮高表达,可能与胆脂瘤骨质破坏有关,并可能与IL-1、TNF-α相互作用,共同参与胆脂瘤骨质破坏过程。     

转化生长因子α在中耳胆脂瘤组织中的表达及意义

目的 :研究转化生长因子 (TGFα)在中耳胆脂瘤中的表达 ,探讨TGFα在胆脂瘤上皮细胞增生中的可能作用。方法 :31例胆脂瘤上皮标本及 10例正常外耳道皮肤组织标本制成石蜡切片 ,应用免疫组化 (S P)染色法检测上述两种标本中TGFα的表达情况 ,并采用多媒体彩色图文分析系统 ,对染色结果进行定量分析。结果 :TGFα阳性表达定位于胞浆 ,胆脂瘤上皮组织均呈中等阳性或强阳性反应 ,2 1例标本呈现从基底层向角质层染色逐渐增强的趋势 ,皮下结缔组织中可见散在的阳性细胞 ,主要为成纤维细胞、单核 巨噬细胞、浆细胞 ,少数为淋巴细胞 ;外耳道皮肤组织的阳性反应集中在表皮层 ,其中 8例标本表现为表皮全层稀疏均一的弱阳性表达。胆脂瘤上皮和正常外耳道表皮的TGFα积分吸光度分别为 2 .4 31± 0 .5 87及 1.4 6 3± 0 .14 7,两者差异有显著性意义(t’ =1.95 ,P <0 .0 5 )。结论 :胆脂瘤组织中TGFα的含量较正常皮肤组织显著增高 ,TGFα可能以自分泌和旁分泌机制参与胆脂瘤上皮增生的调节 ,局部炎症反应可能构成特殊微环境 ,诱发并维持胆脂瘤上皮的高度增生     

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.     

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。     

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。     

肿瘤坏死因子-α在中耳胆脂瘤骨质破坏中的作用

目的 :研究肿瘤坏死因子 - α(TNF- α)在中耳胆脂瘤组织中的表达 ,探讨其在中耳胆脂瘤骨质破坏中的作用。方法 :采用免疫组织化学 SABC染色法和计算机图像定量分析法 ,对 2 5例中耳胆脂瘤和 10例正常外耳道皮肤中 TNF- α的表达情况进行观察。结果 :TNF- α不仅在胆脂瘤上皮的各层细胞胞浆中 ,而且在皮下组织的炎性细胞中都有高度表达。定量分析显示胆脂瘤上皮中 TNF- α的含量显著高于外耳道皮肤 (P <0 .0 5 )。结论 :TNF-α可能是胆脂瘤引起骨质破坏的一个因素。     

PTEN、P-Akt和NF-κB在中耳胆脂瘤上皮中的表达和意义

目的研究10号染色体缺失张力蛋白磷酸酶(phosphatase and tensin homologue deleted on chromosome ten,PTEN)、磷酸化Akt(P-Akt)及核转录因子-κB(NF-κB)在中耳胆脂瘤上皮中的表达,探讨PI3K(phosphatidylinositol-3-kinase,磷脂酰肌醇-3激酶)-Akt信号通路在中耳胆脂瘤上皮细胞过度增殖机制中的可能作用。方法采用免疫组织化学SP法(辣根酶标记链霉卵白素连接法,streptavidin-peroxidase conjugated method)检测30例中耳胆脂瘤组织标本与15例正常外耳道皮肤标本中PTEN、P-Akt及NF-κB蛋白的表达。结果 PTEN蛋白阳性表达主要定位于上皮细胞核,其在中耳胆脂瘤上皮中阳性表达率为36.7%,明显低于正常外耳道皮肤组的93.3%(P<0.01);P-Akt蛋白阳性表达主要定位于上皮细胞胞质,其在中耳胆脂瘤上皮中阳性表达率为70.0%,明显高于正常外耳道皮肤组的26.7%(P<0.01);NF-κB蛋白阳性表达定位于上皮细胞核,其在中耳胆脂瘤上皮中阳性表达率为63.3%,...     

Expression of RANKL and OPG in middle ear cholesteatoma tissue

OBJECTIVES: The objective of this study was to investigate how the expression of the RANK-RANKL-OPG system mediates the formation and differentiation of osteoclasts and causes bone resorption in cholesteatoma. METHODS: An immunohistochemical analysis was carried out in 22 cholesteatoma tissues obtained during middle ear surgery and 15 normal postauricular skin tissues to examine the expression of RANKL and OPG. RESULTS: All 22 cases of cholesteatoma and the 15 cases of normal postauricular skin expressed RANKL and OPG. The count and rate of RANKL-positive cells in cholesteatoma was significantly higher than in normal postauricular skin. The count and rate of OPG-positive cells in normal postauricular skin was significantly higher than in cholesteatoma. The ratio of the positive expression rates of RANKL and OPG in cholesteatoma was statistically higher than in normal postauricular skin. CONCLUSIONS: We provide evidence suggesting that RANKL, which activates osteoclasts, plays a significant role in the mechanism of bone destruction in cholesteatoma, and that the ratio of RANKL to OPG may be a reliable indicator of bone destruction in cholesteatoma.     

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.     

Chromosome 8 aneuploidy in acquired cholesteatoma

OBJECTIVE: To investigate the incidence of chromosome 8 aneuploidy in acquired cholesteatoma. MATERIAL AND METHOD: Cholesteatoma tissue and postauricular skin as a control were surgically obtained from 12 patients with acquired cholesteatoma. Fluorescence in situ hybridization (FISH) analysis using a chromosome 8-specific alpha-satellite DNA probe was performed on the interphase nuclei. Two hundred cells were analyzed for each of the samples. RESULTS: Chromosome 8 aneuploidy was found in 9/12 patients whereas a normal cell structure with 2 signals was observed in the remaining 3 patients. In samples with chromosome 8 aneuploidy, the mean proportion of aneuploidy was 25.6%, including monosomy (3.2%), trisomy (16.1%), tetrasomy (4.9%) and more than tetrasomy (1.4%). The number of aneuploid cells in recurrent cases was more than that in non-recurrent cases. CONCLUSION: A numerical aberration of chromosome 8 was found in patients with acquired cholesteatoma. Our results support the hypothesis that chromosome 8 may be a prognostic factor for cholesteatoma and an indicator in the follow-up of patients with cholesteatoma.     

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。     

Epidermal growth factor in cholesteatoma--the second report: the localization in the horny layer

The localization of epidermal growth factor (EGF) in human cholesteatoma tissue, normal ear drum and external auditory canal skin was examined immunohistochemically, using avidin-biotin peroxidase complex method. Bouin-fixed tissue was stained for investigation of horny layer in the epidermis, because fixation in Bouin's solution provides better preservation of the antigen. In the horny layer of cholesteatoma tissue, 19 out of 24 cases had EGF-positive immunoreactivity (79%). In 2 cases of normal external auditory canal skin, 4 cases of normal ear drum and a case of postauricular skin, no EGF-immunoreactivity was revealed in the horny layer. EGF was assayed in the debris of cholesteatoma and the horny layer of the normal bony external canal with dot blot immunoassay. EGF content of the debris was higher than that of the horny layer of normal skin. The result of the first report suggests the activity of cholesteatoma exists in the subcutaneous tissue (see the previous paper). In this report EGF content of cholesteatoma in the horny layer was found higher than that of normal external skin. This result demonstrates that EGF in the horny layer plays an important role in accelerating the growth and bony destruction in cholesteatoma. To summarize these two reports, the following conclusion was reached. In the epidermis EGF content is equal in cholesteatoma and normal skin. But in the subcutaneous tissue and the horny layer EGF content of cholesteatoma is higher than that of normal skin. EGF in situ may be strongly related to the growth and bony destruction of cholesteatoma.     

Cholesteatoma epithelium is characterized by increased expression of Ki-67, p53 and p21, with minimal apoptosis

OBJECTIVE: To investigate differences in cell proliferation, cell cycle arrest and apoptosis between cholesteatoma and control skin. MATERIAL AND METHODS: Immunohistochemical sections of 15 cholesteatoma and 15 paired control retro-auricular skin samples were examined for Ki-67, p53, p21 and active caspase 3, using image analysis, as well as for DNA fragmentation. RESULTS: The retro-auricular skin samples contained 5.7% +/- 3.6%, Ki-67-positive cells and showed a normal expression pattern. In the cholesteatoma epithelium 11.7% +/- 9.5% of the cells were Ki-67-positive and these cells were dominantly expressed in the basal and parabasal cell layers. Retro-auricular skin contained 5.8% +/- 5.4% p53-positive cells and 1.0% +/- 0.9%, p21-positive cells. In the cholesteatoma epithelium 17.8% +/- 12.3% of the cells were p53-positive and 14.3% +/- 11.6% were p21-positive The expression of Ki-67, p53 and p21 differed significantly between the two groups (p < 0.05). In the cholesteatoma epithelium a positive correlation was found between p53 and p21 expression (p = 0.016). Active caspase 3 positivity and DNA fragmentation were rarely seen in the cholesteatoma epithelium. CONCLUSION: Our results indicate that increased cell proliferation in cholesteatoma epithelium is accompanied by an increase in p53 and p21 protein levels, whilst apoptosis is minimal.     

Expression patterns of Ki-67 and telomerase activity in middle ear cholesteatoma.

BACKGROUND: Keratinocytes in cholesteatoma demonstrate uncoordinated hyperproliferation, migration, and invasion properties. There is a controversy regarding the impact of Ki-67 and telomerase activities on cellular proliferation in cholesteatoma. We studied expression of Ki-67 protein and telomerase activity in cholesteatoma and its relationship with clinical findings. METHODS: The expression level of Ki-67 protein was examined by immunohistochemical analysis of 51 cholesteatomas and 6 skin tissues obtained from patients during ear surgery. Telomerase activity was determined in 23 samples of cholesteatomas and 6 skin samples by polymerase chain reaction-based telomeric-repeat amplification protocol assay. RESULTS: The presence of Ki-67 protein was observed in 21 (41.2%) of 51 samples of acquired cholesteatoma. The average Ki-67 labeling index in the cholesteatoma group was 28.9 +/- 9.2 and was higher than that in the skin group (18.2 +/- 6.1). Telomerase activity was detected in 2 (8.7%) of 23 samples of cholesteatoma (21 of them were Ki-67 staining positive and 2, negative) and in 3 (50%) of 6 of control skin samples (p < 0.05). CONCLUSION: This study showed increased expression of Ki-67 in cholesteatoma, whereas there was no significant difference in rate of Ki-67 positive staining between skin and cholesteatoma (p = 0.066). Telomerase activation is a rare event in cholesteatoma. We assume that the absence of telomerase may lead to generation dysfunctional telomeres what in turn may impair the proliferative capacity of cholesteatoma.