【作者】 张海令；

【导师】 许安廷；

【作者基本信息】 山东大学 ， 临床医学， 2012， 博士

【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(P<0.05)。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(P>0.05)。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(P<0.05),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(P>0.05)。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,P<0.05)；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,P<0.05)。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,P<0.05)；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,P<0.05)。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。更多还原

【Abstract】 Background and Objective:Nasal Polyps (NP) is a common proliferative disease of Rhinology, its pathogenesis is complex, has not really been clarified. Since the widespread of functional endoscopic sinus surgery technology, the recurrence rate is significantly reduced. But part of the nasal polyps, such as nasal polyposis, the recurrence rate after surgery is always high. Some patients have to take low-dose hormone oral preparations for a long time after undergoing multiple surgeries and long-term use of topical corticosteroid hormone nasal spray, and even doing like this, some polyps will still relapse again, has greatly increased the suffering, ideas and economic burden of patients.Individual prognosis of such patients with nasal polyps, is now almost entirely dependent on the clinical experience of Rhinology physician subjective judgments, there is no widely accepted objective indicators of clinical reference value. In recent years, studies on apoptosis in the pathogenesis of nasal polyp is gradually increasing. some fruitful works Have been made. Especially in glandular proliferation/suppressing proliferation or promoting apoptosis/suppression of apoptotic factors in dynamic equilibrium, the polyp epithelium, polyps, eosinophils. These results in preliminarily that the nasal polyp epithelial cell proliferation; apoptotic genes/antiapoptotic gene imbalance, apoptosis inhibition is an important factor in nasal polyps, the eosinophil count increased.Livin is a new member of inhibitors of apoptosis proteins, IAPs. Livin also have the BIR and RING zinc finger domains, ralative to Caspases inhibition mediated apoptosis. It was not expressed in most normal adult tissues, high expression in the tumors and the proliferation of diseased cells. The Caspases protein plays an important position in apoptosis. Necessarily through Caspase cascade reaction, follow the activation and induction of apoptosis. Livin can be of interaction with multiple members of the inhibition of apoptosis protein, for example, Livin can inhibit its function by the combination of caspase-9precursor protein.Smac/DIABLO is a pro-apoptosis-related protein, with a239-amino acid residues, usually located in the mitochondrial membrane gap. When apoptotic signals stimulate Smac/DIABLO and Smac release into the cytoplasm along with cytochrome c, the mature Smac after leaving the mitochondria, the domain BIR3surface groove binding of XIAP, of Livin and other IAPs family members, is for the combination with caspase9, through Smac’s competitive removal of IAPs inhibition of apoptosis, then further activated Caspase cascade reactions that ultimately induce apoptosis.Mcm3, minichromosome maintenance protein3, as an MCMS family, closely related organisms during DNA replication. MCMs family includes seven members. Minichromosome is formed by the yeast nucleosome packaging structure, the structural basis of yeast replication.Compared with the classic proliferation marker Ki-67, although both are located in the proliferating cells. Only Mcm3expressed in the growth of a considerable number of quiescent cells positive contrasted to Ki-67’s negative expression. Mcm3, considered as a cell proliferation marker, is more meaningful than the Ki-67.There is rare report of Livin, Smac and Mcm3expression in NP research. Combined detection and analysis, and make the correlation analysis with NP clinical classification stage and postoperative recurrence rate, and to be worked as meaningful referrence indexes for the otolaryngologist, and to make more appropriate individualized surgical treatment and follow-up program.Objective of This research is to explore the apoptosis mechanism of nasal polyps’ pathogenesis via detecting the expressions of two related factors of Apoptosis:Livin and Smac, and Mcm3, a kind of index protein which is sensitive to multiplication of human cells, in human nasal polyps (NP). Furthermore, by investigating the relationships with NP’s different clinical stages and recurrence rate, to find a reliable, molecular level’s approach by which ENT doctor can prejudge the possibility of postoperative recurrence, and may be applied as a referrence index for ENT doctors to make the plan of follow up and treatment.Methods:Choosing randomly80cases from the preserved blocks of nasal polyps patients who were followed at least12months after FESS operation.12cases of normal uncinate process mucosa were served as contrast. With polyclonal anti-human Livin, monoclonal anti-human Smac and monoclonal anti-human Mcm3, immunohistochemistry SP method was used to examine the expression of Livin,Smac and Mcm3in every case. Beige or puce granules appearing in NP cells were regarded as positive signal. Every case was observed randomly for ten eyeshots (microscope X400). The results were judged by semi-quantificational integral standard. All the data were disposed by statistics.Results:Of the80NP cases, Livin positive were54, positive rate was66.7%(54/80),cytoplasm coloration, the main location was in gland epithelium cells. Of the contrast, Livin positive was none. Mcm3positive were46, positive rate was56.7%(46/80), caryon coloration, the main location was in the epidermis cells of polyps. Two of the contrast was positive, positive rate was33.3%(4/12). Smac positive were42, positive rate was52.5%(42/80), cytoplasm coloration, the main location was in the epidermis cells of polyps. All of the contrast were positive, positive rate was91.7%(11/12). The expressive intensity of Livin、Smac between NP and normal mucosa were statistical different (P<0.05). Contrarily, there was no statistical difference in the expressive intensity of Mcm3between NP and normal mucosa (P>0.05). The expressive intensity of Livin and Smac between different clinical classifications of NP showed statistical difference (P<0.05). However, the expressive intensity of Mcm3between different classifications of NP didn’t have statistical difference (P>0.05).There was a positive correlation between co-expressive intensity of Livin and Mcm3;and NP classifications differentiated by type and stage (R’s=0.453, P<0.05). Being completely contrary, for Smac, it was a negative correlation (R’s=-0.429, P<0.05).Furthermore, between the co-expressive intensity of Livin and Mcm3and the relapse rate of postoperative patients, a positive linear correlation was presented (R=0.9569, P<0.05).For Smac, it showed a negative linear correlation (R=-0.9781, P<0.05)Conclusion:Livin plays an important role in the genesis and development of the glands of NP. There is an obvious Smac down regulation in NP epidermis cells. In the pathogenesis of NP, the heightening of cell proliferative potential of NP probably acts as a subordinate role, the principal factor is that normal apoptosis is restrained. There is a positive correlation between the classifications of NP and the depressed extent of apoptosis. Detecting Livin、Smac and Mcm3synchronously can be considered as a reliable reference index by which we can forecast the probability of postoperative recurrence of individual NP patient. Objective:To explore the apoptosis mechanism of nasal polyps’ pathogenesis via detecting the expressions of two related factors of Apoptosis:Livin and Smac, and Mcm3, a kind of index protein which is sensitive to multiplication of human cells, in human nasal polyps (NP). Furthermore, by investigating the relationships with NP’s different clinical stages and recurrence rate, to find a reliable, molecular level’s approach by which ENT doctor can prejudge the possibility of postoperative recurrence, and may be applied as a referrence index for ENT doctors to make the plan of follow up and treatment.Methods:Choosing randomly80cases from the preserved blocks of nasal polyps patients who were followed at least12months after FESS operation.12cases of normal uncinate process mucosa were served as contrast. With polyclonal anti-human Livin, monoclonal anti-human Smac and monoclonal anti-human Mcm3, immunohistochemistry SP method was used to examine the expression of Livin, Smac and Mcm3in every case. Beige or puce granules appearing in NP cells were regarded as positive signal. Every case was observed randomly for five eyeshots (microscope×400). The results were judged by semi-quantificational integral standard. All the data were disposed by statistics. Results:Of the80NP cases, Livin positive were54, positive rate was66.7%(54/80), cytoplasm coloration, the main location was in gland epithelium cells. Of the contrast, Livin positive was none. Mcm3positive were46, positive rate was56.7%(46/80), caryon coloration, the main location was in the epidermis cells of polyps. Two of the contrast was positive, positive rate was16.7%(2/12). Smac positive were42, positive rate was52.5%(42/80),cytoplasm coloration, the main location was in the epidermis cells of polyps. All of the contrast were positive, positive rate was91.7%(11/12). The expressive intensity of Livin、Smac between NP and normal mucosa were statistical different (P<0.05). Contrarily, there was no statistical difference in the expressive intensity of Mcm3between NP and normal mucosa (P>0.05).The expressive intensity of Livin and Smac between different clinical classifications of NP showed statistical difference(P<0.05). However, the expressive intensity of Mcm3between different classifications of NP didn’t have statistical difference (P>0.05). There was a positive correlation between co-expressive intensity of Livin and Mcm3;and NP classifications differentiated by type and stage (R’s=0.606, P<0.05). Being completely contrary, for Smac, it was a negative correlation (R’s=-0.628P<0.05). Furthermore, between the co-expressive intensity of Livin and Mcm3and the relapse rate of postoperative patients, a positive linear correlation was presented (R=0.9588, t=4.7779, P<0.05). For Smac, it showed a negative linear correlation (R=-0.9992, t=33.30, P<0.05)Conclusions:Livin plays an important role in the genesis and development of the glands of NP. There is an obvious Smac down regulation in NP epidermis cells. In the pathogenesis of NP, the heightening of cell proliferative potential of NP probably acts as a subordinate role, the principal factor is that normal apoptosis is restrained. There is a positive correlation between the classifications of NP and the depressed extent of apoptosis. Detecting Livin、Smac and Mcm3synchronously can be considered as a reliable reference index by which we can forecast the probability of postoperative recurrence of individual NP patient.更多还原