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。     

表皮生长因子受体和Ki67及p16在成人中耳胆脂瘤中的表达

目的:研究表皮生长因子受体(EGFR)、Ki67、p16在成人中耳继发性胆脂瘤上皮中的表达情况,分析它们之间的相互关系,探讨其表达对胆脂瘤上皮侵袭能力的影响。方法:应用免疫组织化学SP染色方法检测EGFR、Ki67和p16在30例成人中耳胆脂瘤上皮、21例成人胆脂瘤患者外耳道正常皮肤、17例正常人外耳道中的表达情况,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:EGFR、Ki67、p16在成人中耳继发性胆脂瘤上皮中阳性表达率分别为70.0%,60.0%,46.7%,与外耳道正常皮肤相比表达均差异有统计学意义。成人中耳胆脂瘤中EGFR、Ki67与p16之间表达均无相关性(均P>0.05)。胆脂瘤侵袭能力与EGFR、Ki67表达有显著相关性(均P<0.01)。EGFR、Ki67表达灰度值越低,表达密度越高,胆脂瘤侵袭能力越强。p16在成人中耳胆脂瘤中的表达与侵袭能力之间无相关性(P>0.05)。EGFR、Ki67、p16在成人中耳胆脂瘤中阳性细胞主要分布于上皮全层,以基底层和棘层为著,呈高度表达;而在对照组中阳性细胞仅在基底层表达,呈弱表达。结论:EGFR、Ki67、p16在成人中耳胆脂瘤中呈高表达,EGFR、Ki67的表达与成人中耳胆脂瘤的侵袭能力有高度相关性,提示成人中耳胆脂瘤具有高度增殖能力,其中细胞因子EGFR、Ki67、p16起到重要的作用。     

核转录因子κ-Bp65在中耳胆脂瘤上皮中的表达和意义

目的检测核转录因子-κBp65（nuclear factor kappa Bp65,NF-κBp65）在中耳胆脂瘤上皮中的表达和活化,探讨其在中耳胆脂瘤发病机制中的可能作用。方法采用免疫组织化学SP法检测30例中耳胆脂瘤组织标本与15例正常外耳道皮肤标本中NF-κBp65蛋白的表达。结果NF-κBp65蛋白阳性表达定位于上皮细胞核。NF-κBp65蛋白在中耳胆脂瘤上皮组织中阳性表达率为63.3%,明显高于正常外耳道皮肤组的20.0%（P〈0.01）。结论NF-κBp65蛋白在中耳胆脂瘤上皮的异常表达可能在胆脂瘤的发生、发展过程中起重要作用。胆脂瘤上皮中NF-κBp65的活化可能参与了胆脂瘤上皮细胞过度增殖机制。     

PTEN、 P-Akt和NF-кB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- kB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％,明显高于正常外耳道皮肤组的20.0％ (P＜ 0.01).在30例中耳胆脂瘤上皮组织中,PTEN分别与P-Akt、NF-κB蛋白的表达之间呈显著负相关(P＜0.01),而P-Akt和NF- κB蛋白的表达呈显著正相关(P＜0.01).结论 PTEN、P-Akt和NF- κB在中耳胆脂瘤上皮的异常表达可能在胆脂瘤的发生、发展过程中起重要作用.胆脂瘤上皮中PI3K-Akt信号通路的激活可能参与了胆脂瘤上皮细胞过度增殖机制.     

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 (epidermalgrowthfactorreceptor,EGFR) ,增殖细胞核抗原Ki6 7、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Ki6 7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 ,为连续的棕黄色线状和 (或 )带状图案。 4例胆脂瘤上皮基膜下结缔组织中的血管比皮肤丰富。结论　EGFR和Ki6 7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     

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。     

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…     

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。     

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%,...     

角化细胞生长因子及受体在鼓膜穿孔邻近皮肤的研究

目的观察鼓膜穿孔部位邻近皮肤中角化细胞生长因子（keratinocyte growth factor,KGF）及其受体角化细胞生长因子受体（keratinocyte growth factor receptor,KGFR）的表达情况,分析其在慢性化脓性中耳炎不同转归中的作用。方法应用免疫组化SP染色方法和多媒体图像分析系统,观察20例继发性胆脂瘤型中耳炎鼓膜穿孔部位邻近皮肤、胆脂瘤上皮以及耳道深部正常皮肤的KGF和KGFR表达,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鼓膜穿孔邻近皮肤KGF和KGFR的阳性表达率分别为（33.135±6.364）%和（19.965±10.570）%,介于胆脂瘤上皮与耳道正常皮肤之间,明显高于非胆脂瘤型中耳炎患者相应部位（19.380±2.827）%和（13.145±7.935）%。结论KGF和KGFR在慢性化脓性中耳炎不同部位的表达依次上升,在胆脂瘤型中耳炎鼓膜穿孔邻近皮肤的表达明显高于非胆脂瘤型中耳炎,说明胆脂瘤型中耳炎该处皮肤增生更活跃。     

Expression of keratinocyte growth factor (KGF) and its receptor in a middle-ear cavity problem

Objectives

To investigate the pathogenesis of one of the most troublesome conditions following ear surgery, a middle-ear cavity problem.

Methods

Keratinocyte growth factor (KGF) and its receptor (KGFR), the ratio of proliferating epithelial cells using Ki-67, and the extent of infiltration of B cells and T cells were examined immunohistochemically in 10 ears with a cavity problem, 70 ears with cholesteatoma and 8 ears with normal skin at the retroauricular incision.

Results

KGF was positive in 40% of cavity problem specimens, 37.5% of normal skin specimens, and was positive in 88% of cholesteatoma specimens (cavity problem vs. cholesteatoma, p = 0.0004). The positive rate of KGFR in the cavity problem group (33.3%) was between those in cholesteatoma (60%) and normal skin (0%). In contrast to the cholesteatoma specimens, a significantly smaller number of Ki-67 labeling index (LI) was detected in the cavity problem specimens. B cell LI was significantly higher but T cell LI was significantly lower in the cavity problem specimens than in the cholesteatoma group.

Conclusions

Our present study indicated that the discordance of paracrine action between stromal KGF and epithelial KGFR with a large number of infiltrating B cells may play an important role in the pathogenesis of a cavity problem.     

Keratinocyte Growth Factor (KGF) Modulates Epidermal Progenitor Cell Kinetics through Activation of p63 in Middle Ear Cholesteatoma

The basal stem/progenitor cell maintains homeostasis of the epidermis. Progressive disturbance of this homeostasis has been implicated as a possible cause in the pathogenesis of epithelial disease, such as middle ear cholesteatoma. In many cases of stem/progenitor cell regulation, the importance of extracellular signals provided by the surrounding cells is well-recognized. Keratinocyte growth factor (KGF) is a mesenchymal-cell-derived paracrine growth factor that specifically participates in skin homeostasis; however, the overexpression of KGF induces middle ear cholesteatoma. In this study, two kinds of thymidine analogs were transferred at different time points and we investigated the effects of overexpressed KGF on the cell kinetics of stem/progenitor cells in vivo. As a result, BrdU(+)EdU(+) cells (stem/progenitor cells) were detected in the thickened epithelium of KGF-transfected specimens. The use of a high-resolution microscope enabled us to analyze the phosphorylated level of p63 in individual nuclei, and the results clearly demonstrated that BrdU(+)EdU(+) cells are regarded as progenitor cells. In the overexpression of KGF, the stimulation of progenitor cell proliferation was inhibited by SU5402, an inhibitor for tyrosine kinase of KGFR. These findings indicate that KGF overexpression may increase stem/progenitor cell proliferation and block terminal differentiation, resulting in epithelial hyperplasia, which is typical in middle ear cholesteatoma.     

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     

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.     

Roles of Cytokines and Cell Cycle Regulating Substances in Proliferation of Cholesteatoma Epithelium

Objectives: It can be surmised that the cell cycle must be involved in cell proliferation of the epithelium of middle ear cholesteatoma. Thus a comparative study was conducted of the levels of expression of cyclin-dependent kinase 2 (cdk2) and cyclin-dependent kinase 4 (cdk4)—substances known to be involved in the cell cycle—in cholesteatoma epithelium and the normal epithelium of the bony region of the external ear canal. In addition, it has been reported that the expression of cytokines in the epithelium is accelerated in response to subepithelial inflammation. This suggests that an interaction between the epithelium and subepithelium, which is subject to paracrine regulation, is deeply involved in epithelial proliferation. Accordingly, attention was focused on interleukin?1α (IL-1α) and keratinocyte growth factor (KGF), cytokines which are found in the subepithelium, and experiments were conducted to elucidate their effects on the expression of the substances known to be involved in the cell cycle. Methods: The expressions of cdk2 and cdk4 in the cholesteatoma epithelium and external ear canal epithelium were investigated by an immunohistochemical technique. In addition, cultured human keratinocytes were grown in medium containing IL-1α or KGF at concentrations of 0, 20, and 100 ng/mL, and the differences in the expression of cdk2 and cdk4 were investigated and compared by Western blot analysis. Results: In the cholesteatoma epithelium specimens, cdk2 and cdk4 were observed to be expressed in the basal and parabasal layers and in the upper layer (prickle layer and granular layer). Their expression tended to be increased compared with their expression in the normal external ear canal epithelium, and this tendency was marked in subepithelial sites showing severe inflammation. In addition, exposure of cultured human keratinocytes to IL-1α or KGF resulted in accelerated expression of both cdk2 and cdk4, and this was especially striking in the case of addition of KGF. Conclusion: It can be surmised that, in cholesteatoma, accelerated expression of IL-1α and KGF by inflammatory cells at subepithelial sites of inflammation leads to upregulation of cdk2 and cdk4 in epithelial cells and to cell proliferation. It was concluded that this is at least one sequence of events involved in the mechanism causing epithelial proliferation in cholesteatoma.     

Topical application of 5-fluorouracil on attic cholesteatoma results in downregulation of keratinocyte growth factor and reduction of proliferative activity

To investigate the cell-biological effect of topically applied 5-fluorouracil (5-FU) on middle ear cholesteatoma, 12 attic cholesteatomas were treated with topical application of 5-FU cream, two to five times with an interval of 2 weeks (5-FU group). The control group comprised 65 cholesteatoma that were not treated with 5-FU. All lesions were later excised surgically and processed for immunohistochemical analyses of Ki-67, keratinocyte growth factor (KGF) and its receptor (KGFR). 5-FU significantly reduced the expression of KGF, did not change KGFR expression, and significantly reduced the Ki-67 labeling index, relative to the control group. The effect of 5-FU on cholesteatoma seems to be mediated, at least in part, through downregulation of KGF in stromal cells and reduction of the proliferative activity of epithelial cells.     

Angiogenesis and angiogenic growth factors in middle ear cholesteatoma

HYPOTHESIS: This study aimed to analyze the localization and distribution of vessels and of these angiogenic growth factors: basic fibroblast growth factor (FGF-2), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF) in middle ear cholesteatoma in comparison with normal middle ear mucosa and auditory meatal skin. BACKGROUND: Angiogenesis is particularly important in many normal and pathologic processes, including wound healing and inflammation. Because proliferating tissues require an enhanced blood supply, angiogenesis appears to be a prerequisite for the expansion of cholesteatoma. METHODS: The expression of FGF-2, TGF-alpha, TGF-beta1, and VEGF was studied by immunohistochemistry. The amount of vessels (collagen type IV staining) was determined by an automatic imaging analyzing system. RESULTS: The results showed an altered expression and distribution of VEGF, FGF-2, TGF-alpha, and TGF-beta1 in cholesteatoma in relation to middle ear mucosa and auditory meatal skin. The results were consistent with rapidly growing, activated keratinocytes and stromal cells. Vascularization within the perimatrix of cholesteatoma showed a 4.3-fold increase compared with middle ear mucosa and a twofold increase compared with ear canal skin. An increase of 3.2- to 4-fold in the number of vessels was observed. A close relationship was seen between the density of capillaries, degree of inflammation, and expression of the angiogenic factors investigated, and an increased number of microvessels in cholesteatoma tissue. CONCLUSIONS: Angiogenesis enables and supports the sustained migration of keratinocytes into the middle ear cavity. Therefore, it is a pivotal factor in the destructive behavior of middle ear cholesteatoma.