鼻息肉中LIVIN，SMAC及MCM3的表达及与其临床分型分期和复发率的相关性分析

发布时间：2018-05-17 01:28

本文选题：鼻息肉 + 细胞凋亡　；参考：《山东大学》2012年博士论文

【摘要】：研究背景及目的：鼻息肉(Nasal Polyps, NP)是一种常见的鼻科增生性疾病,其发病机制复杂,至今没有真正阐明。自从内窥镜手术广泛开展以来,其术后复发率已明显降低。但部分鼻息肉,如“鼻息肉病”,其手术后的复发率始终较高。有些患者往往在经历多次手术以及长期使用局部皮质类固醇激素喷鼻剂,甚至部分患者须服用较长时间的低剂量激素口服制剂后,仍然会再次复发息肉,大大增加了患者的痛苦及思想与经济负担。对此类鼻息肉患者个体的预后,目前几乎完全依赖鼻科医师的临床经验进行主观判断,尚无广泛认可的具有临床参考价值的客观指标。近年来,在鼻息肉发病机制中关于细胞凋亡方面的研究正在逐步增加。目前已做了一些卓有成效的工作。特别是在腺体促增殖/抑增殖或促凋亡/抑凋亡因素的动态平衡方面、息肉上皮、息肉组织中嗜酸粒细胞等方面均取得一些成果。目前研究结果初步认为鼻息肉上皮细胞有显著的增殖特性；凋亡基因/抑凋亡基因失衡,使细胞凋亡受抑制是鼻息肉组织中嗜酸粒细胞数增多的重要因素之一 Livin是IAPs(inhibitors of apoptosis proteins, IAPs)蛋白家族的新成员,有BIR和RING锌指结构域,能够与Caspases蛋白结合,抑制其介导的细胞凋亡。它在大多数正常成人组织中不表达、在一些肿瘤及增殖性病变细胞中有高表达。Caspases蛋白在介导细胞凋亡中处于重要地位,大多数的刺激性信号是经由Caspase级联反应,激活并诱导细胞凋亡的。Livin与其中多个成员相互作用,抑制细胞凋亡的执行,例如,Livin可结合caspase-9的前体蛋白及其激活的形式,从而抑制其功能。 Smac/DIABLO是一个促进凋亡的重要相关蛋白,具有239-氨基酸残基,一般定位于线粒体的膜间隙。当凋亡信号刺激Smac/DIABLO时,Smac会伴随着细胞色素c的释放进入胞浆,成熟的Smac在离开线粒体后,可与XIAP、Livin等IAPs家族成员锌指结构域BIR3上的表面沟结合,而这个沟是用于结合caspase9的部位,因而Smac能竞争性去除IAPs对凋亡的抑制,从而进一步激活Caspase级联反应,最终诱导细胞凋亡。 Mcm3,即微小染色体维持蛋白3,作为Mcms家族中重要的一员,与生物体DNA复制过程密切相关。MCMs家族目前有7种。微小染色体是指酵母中一系列核小体被包装后形成的结构,是酵母复制的结构基础。同经典的增殖性标记物Ki-67相比,两者虽都定位于增殖细胞中。只有Mcm3能够在相当数量的处于生长静止期的细胞中表达阳性,而Ki-67无表达,可以认为Mcm3作为一种细胞增殖标记物,较Ki-67更有意义。目前关于Livin, Smac和Mcm3在NP中表达的相关研究很少。三者联合检测分析,并与NP临床分型分期和术后复发率之间做相关性研究,指导耳鼻喉科医师制定更加适宜的个体化手术治疗及随访方案,在国内外目前的检索文件中尚未见报道。本文主要研究目的在于：(1)通过联合检测3种典型的细胞凋亡／增殖相关因子：Livin, Smac和Mcm3在NP的表达特点,进一步探索NP在细胞凋亡途径可能的发病机制,为进一步阐明细胞凋亡机制在NP的发生、发展中的重要作用,具有较强的理论意义。(2)并通过对上述3种细胞凋亡／增殖因子的表达水平与NP临床分型分期及术后复发率进行相关性分析,探索一条能较为客观科学地预测NP患者术后复发可能性的分子水平途径,可作为临床参考指标,与鼻科医师的临床经验性判断互为补充,用于指导耳鼻喉科医生对NP患者在手术治疗、围手术期处理以及随访等方面进行个体化的制定,以改善目前主要凭医师临床经验做出判断的现状,有效减少复发,具备实际的临床应用价值。方法：于山东大学第二医院病理科检验并保存的2005年6月～2009年6月期间入院的单纯鼻息肉患者蜡块中随机选取80例,男性52例,女性28例,平均年龄34.5岁(19-57岁),临床分型分期为Ⅱ型Ⅰ期24例,Ⅱ型Ⅱ期26例,Ⅱ型Ⅲ期16例,Ⅲ型14例。均病理确诊为鼻息肉.通过详细询问病史、临床症状、鼻内窥镜检查、变应原皮肤试验(参照2008年WHO制定的《过敏性鼻炎的处理及其对哮喘的影响》以及中华医学会呼吸病学分会哮喘学组制定的2008年《支气管哮喘防治指南》),均排除变应性鼻炎、哮喘等变态反应性疾病史。80例患者随访复查期均超过12个月。随访期内经病理证实复发者(所有复发患者均首先在鼻内窥镜复查时发现息肉样新生物,向患者讲明目前病情并由本人签署我院特殊检查治疗知情同意书后,然后行内窥镜下鼻息肉清理并送病理科再次诊断),Ⅱ型Ⅰ期2例,Ⅱ型Ⅱ期6例,Ⅱ型Ⅲ期6例,Ⅲ型8例。正常对照组：12例,取自2008年5月～2009年12月间我院外伤性脑脊液鼻漏患者手术修补过程中,为正常钩突粘膜,均排除鼻-鼻窦炎及变应性鼻炎、哮喘等变态反应性疾病史(排除方法同鼻息肉组,并结合鼻窦CT)。通过免疫组化SP方法,检测标本组织中Livin, Smac及Mcm3的表达,以胞浆和/或胞核中出现棕黄色或棕褐色颗粒为阳性信号,应用Olympus显微镜和Micro-image图像处理系统,每例标本选取3张切片,每张切片均采用双盲法随机观察10个高倍视野(显微镜下×400),用半定量积分法判断结果,所有数据经统计学处理。结果：80例标本中,Livin表达阳性片为54例,阳性率为66.7%(54/80),主要表达在息肉组织内增生的腺体/腺管上皮细胞,定位于胞浆；对照组12例正常粘膜无1例阳性(0/12)。Mcm3表达阳性片为46例,阳性率为56.7%(46/80),主要表达在息肉上皮细胞,定位于胞核。对照组正常粘膜4例阳性,阳性率为33.3%(4/12),定位于粘膜上皮细胞的胞核。Smac表达阳性为42例,阳性率为52.5%(42/80),主要表达在息肉上皮细胞,部分腺体上皮亦可见阳性表达,定位于胞浆。对照组正常粘膜11例阳性,阳性率91.7%(11/12),主要表达在粘膜上皮细胞,定位于胞浆。Livin,Smac在鼻息肉与正常粘膜对照组之间的表达差异具有统计学意义(P0.05)。Mcm3在鼻息肉与正常粘膜对照组之间的表达差异不具有统计学意义(P0.05)。Livin, Smac表达强度在鼻息肉不同临床分型分期之间具有统计学上的差异(P0.05),可以认为鼻息肉不同临床分型分期中Livin, Smac表达强度是不同的。Mcm3表达强度在鼻息肉不同临床分型分期之间不具有统计学上的差异(P0.05)。Livin、Mcm3联合表达强度与鼻息肉临床分型分期之间呈正相关关系(R's=0.453,P0.05)；而Smac表达强度与鼻息肉临床分型分期之间呈负相关关系(R's=-0.429,P0.05)。Livin、Mcm3联合表达强度与鼻息肉术后复发率之间呈直线正相关关系(R=0.9569,P0.05)；而Smac表达强度与鼻息肉术后复发率之间呈直线负相关关系(R=-0.9781,P0.05)。结论：(1)Livin过度表达可能在鼻息肉内腺体发生发展的过程中具有重要作用。(2)鼻息肉中存在明显的Smac表达受阻。(3)Mcm3标志的鼻息肉细胞增殖活性的提高在其发病机制中可能处于一个相对次要的地位,而凋亡受抑制可能起首要作用。(4)鼻息肉临床分型分期高低与凋亡受抑程度之间存在正相关关系,其分型分期越高,细胞凋亡可能越弱。(5)Livin, Smac及Mcm3作为在细胞凋亡/增殖动态平衡中具有不同作用机理的三个重要因子,其联合检测结果可作为有临床意义的客观参考指标,在分子水平上预测个体鼻息肉患者术后复发的可能性；与临床经验相结合,使耳鼻喉科医师对鼻息肉患者可采取更适宜的个体化手术方案、围手术期处理及随访。
[Abstract]:Background and objective: Nasal Polyps (NP) is a common hyperplastic disease of the nose. Its pathogenesis is complex and has not been clarified so far. Since endoscopic surgery has been widely carried out, the recurrence rate has decreased obviously. However, some nasal polyps, such as "nasal polyposis", have a higher recurrence rate. People often undergo multiple operations and use local corticosteroid nasal spray for a long time, and even some patients have to take a long period of low dose of hormone oral preparation, still relapse polyps, greatly increasing the patient's pain and ideological and economic burden.
The individual prognosis of this kind of nasal polyp is almost entirely dependent on the clinical experience of the physicians, and there is no widely recognized objective index with clinical reference value. In recent years, the research on cell apoptosis in the pathogenesis of nasal polyps is gradually increasing. In particular, some results have been achieved in the aspects of gland proliferation / inhibition or apoptosis / apoptosis / anti apoptosis factors, polypoid epithelium, eosinophils in polyp tissues and other aspects. The results of the present study suggest that the epithelial cells of nasal polyps have significant proliferation characteristics, and the imbalance of apoptotic genes / anti apoptotic genes causes apoptosis to be suppressed. It is one of the important factors in increasing the number of eosinophils in nasal polyps.
Livin is a new member of the IAPs (inhibitors of apoptosis proteins, IAPs) protein family, with BIR and RING zinc finger domains, which can bind to Caspases proteins and inhibit its mediated apoptosis. It is not expressed in most normal adult tissues, and is highly expressed in some tumor and proliferative cells by the high expression of.Caspases protein in mediating cells In apoptosis, most of the stimulant signals are mediated by Caspase cascade, which activates and induces apoptosis of.Livin to interact with multiple members to inhibit the execution of apoptosis. For example, Livin can inhibit its function by combining the precursor protein of caspase-9 and its activated form.
Smac/DIABLO is an important related protein to promote apoptosis, with 239- amino acid residues and generally located in the membrane space of mitochondria. When the apoptotic signal stimulates Smac/DIABLO, Smac will accompany the release of cytochrome c into the cytoplasm. The mature Smac can be separated from the mitochondria and can be with XIAP, Livin and other IAPs family members of the zinc finger domain BIR3. Surface furrows are combined, and the trench is used to bind to caspase9, so Smac can competitively remove the inhibition of apoptosis by IAPs, which further activates the Caspase cascade reaction and eventually induces apoptosis.
Mcm3, a small chromosome maintenance protein 3, as an important member of the Mcms family, is closely related to the DNA replication process in the organism and the.MCMs family currently has 7 species. Small chromosomes refer to the structure formed after a series of nucleosomes are packaged in yeast and the structural basis for yeast replication.
Compared with the classical proliferative marker Ki-67, both of them are located in the proliferating cells. Only Mcm3 can be expressed in a considerable number of cells in the growth stationary phase, while Ki-67 is not expressed. It is considered that Mcm3 is a cell proliferation marker, which is more meaningful than Ki-67.
There are few related studies on the expression of Livin, Smac and Mcm3 in NP. The three joint detection and analysis, and the correlation between the clinical classification and postoperative recurrence rate of NP, guide the Department of ENT physicians to develop a more suitable individualized surgical treatment and follow-up scheme, and have not yet been reported in the current retrieval documents at home and abroad.
The main purpose of this study is: (1) through the joint detection of 3 typical apoptosis / proliferation related factors: Livin, Smac and Mcm3 in the expression of NP, further explore the possible pathogenesis of NP in the apoptosis pathway, in order to further clarify the mechanism of apoptosis in the development of the important role in the development of NP, has a strong theory. (2) through the correlation analysis of the expression level of the 3 kinds of apoptotic / proliferative factors, the clinical classification and the recurrence rate of NP and the recurrence rate after operation, we explore a molecular level way to predict the recurrence possibility of NP patients objectively and scientifically, which can be used as the clinical reference index and the clinical empirical judgment of the physicians. Complementary to each other, it is used to guide doctors in Department of ENT to make individualized formulation of NP patients in the aspects of surgical treatment, perioperative management and follow-up, in order to improve the current status of judgment mainly based on clinical experience of doctors, reduce recurrence effectively, and have practical clinical value.
Methods: 80 cases were randomly selected from the paraffin block of patients with simple nasal polyps from June 2005 to June 2009 of the second hospital of Shandong University. 52 males and 28 females, with an average age of 34.5 years (19-57 years), were divided into stage I stage 24, II stage II 26, type II 16 and 14. 14 cases. The medical history, clinical symptoms, nasal endoscopy, allergen skin test (referring to the treatment of allergic rhinitis and its impact on asthma by the 2008 WHO), and the 2008 < guidelines for the prevention and control of bronchial asthma from the asthma group of the Chinese Medical Association for respiratory diseases), were all excluded. .80 patients with asthma, such as asthma, were followed up for more than 12 months. During the follow-up period, the recurrent patients (all the recurrent patients first found the polypoid neoplasm at the time of the endoscopic review). There were 2 cases of stage II, 6 cases of stage II, 6 cases of stage II, and 8 cases of type III.
The normal control group: 12 cases, from May 2008 to December 2009, during the surgical repair of traumatic cerebrospinal rhinorrhea in our hospital, the normal mucous membrane of the uncinate process, the history of allergic diseases such as rhinosinusitis and allergic rhinitis, asthma were excluded (the method was with the nasal polyp group and combined with the CT of the paranasal sinus).
The expression of Livin, Smac and Mcm3 in specimen tissues was detected by immunohistochemical SP method. The positive signals of brown yellow or brown brown granules in cytoplasm and / or nucleus were detected by Olympus microscope and Micro-image image processing system. 3 slices were selected for each specimen, and 10 high times of visual field were observed by double blind method in each slice. Microscopically * 400, the results were determined by semi quantitative integration, and all data were processed statistically.
Results: of the 80 specimens, 54 cases of Livin positive expression were positive, the positive rate was 66.7% (54/80). The gland / glandular epithelial cells, which were mainly expressed in the polyp tissues, were located in the cytoplasm. In the control group, there were no 1 positive (0/12) positive (0/12).Mcm3 positive tablets in 12 normal mucosa, and the positive rate was 56.7% (46/80), mainly expressed in polyp epithelial cells. The positive rate of 4 cases of normal mucosa in the control group was 33.3% (4/12). The positive rate of.Smac expression in the epithelial cells of the mucosa was 42, the positive rate was 52.5% (42/80). The positive expression was mainly expressed in the epithelial cells of polyps, and the positive expression was found in some glandular epithelium. In the control group, 11 cases of normal mucous membrane were positive, the positive rate was 91.7% (11/12 The expression of.Livin and Smac between nasal polyps and normal mucosa was statistically significant (P0.05) and there was no statistical difference between nasal polyps and normal mucosa (P0.05).Livin (P0.05), and the expression intensity of Smac in nasal polyps was different in different clinical types of nasal polyps. There was a statistically significant difference (P0.05) between the different stages of nasal polyps (Livin) and the expression intensity of Smac was not statistically significant (P0.05).Livin between the different clinical types and stages of nasal polyps (P0.05), and the joint expression intensity of Mcm3 and the clinical classification of nasal polyps were Cheng Zhengxiang.Livin. There was a negative correlation between the expression intensity of Smac and the clinical classification and staging of nasal polyps (R's=-0.429, P0.05).Livin. There was a positive linear correlation between the joint expression intensity of Mcm3 and the recurrence rate after the operation of nasal polyps (R=0.9569, P0.05), while the intensity of Smac was negatively correlated with the recurrence rate of nasal polyps. The Department (R=-0.9781, P0.05).
Conclusion: (1) overexpression of Livin may play an important role in the development of glands in nasal polyps. (2) there is an obvious obstruction of Smac expression in nasal polyps. (3) the enhancement of proliferation activity of nasal polyps in Mcm3 markers may be in a relatively minor position in its pathogenesis, and the inhibition of apoptosis may play a primary role. (4) there is a positive correlation between the clinical classification of nasal polyps and the inhibition of apoptosis, the higher the classification stage, the weaker the apoptosis may be. (5) Livin, Smac and Mcm3 are the three important factors that have different mechanisms of action in the dynamic balance of cell apoptosis / proliferation, and the combined detection results can be used as objective references for clinical significance. The test index, at the molecular level, predicts the possibility of postoperative recurrence of individual nasal polyps. Combined with clinical experience, Department of ENT physicians can take a more appropriate individualized operation scheme for patients with nasal polyps, perioperative management and follow-up.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R765.25

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