本文選題：鼻息肉 + 細(xì)胞凋亡��； 參考：《山東大學(xué)》2012年博士論文

【摘要】：研究背景及目的：鼻息肉(Nasal Polyps, NP)是一種常見的鼻科增生性疾病,其發(fā)病機(jī)制復(fù)雜,至今沒有真正闡明。自從內(nèi)窺鏡手術(shù)廣泛開展以來,其術(shù)后復(fù)發(fā)率已明顯降低。但部分鼻息肉,如“鼻息肉病”,其手術(shù)后的復(fù)發(fā)率始終較高。有些患者往往在經(jīng)歷多次手術(shù)以及長期使用局部皮質(zhì)類固醇激素噴鼻劑,甚至部分患者須服用較長時(shí)間的低劑量激素口服制劑后,仍然會(huì)再次復(fù)發(fā)息肉,大大增加了患者的痛苦及思想與經(jīng)濟(jì)負(fù)擔(dān)。 對(duì)此類鼻息肉患者個(gè)體的預(yù)后,目前幾乎完全依賴鼻科醫(yī)師的臨床經(jīng)驗(yàn)進(jìn)行主觀判斷,尚無廣泛認(rèn)可的具有臨床參考價(jià)值的客觀指標(biāo)。近年來,在鼻息肉發(fā)病機(jī)制中關(guān)于細(xì)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。特別是在腺體促增殖/抑增殖或促凋亡/抑凋亡因素的動(dòng)態(tài)平衡方面、息肉上皮、息肉組織中嗜酸粒細(xì)胞等方面均取得一些成果。目前研究結(jié)果初步認(rèn)為鼻息肉上皮細(xì)胞有顯著的增殖特性；凋亡基因/抑凋亡基因失衡,使細(xì)胞凋亡受抑制是鼻息肉組織中嗜酸粒細(xì)胞數(shù)增多的重要因素之一 Livin是IAPs(inhibitors of apoptosis proteins, IAPs)蛋白家族的新成員,有BIR和RING鋅指結(jié)構(gòu)域,能夠與Caspases蛋白結(jié)合,抑制其介導(dǎo)的細(xì)胞凋亡。它在大多數(shù)正常成人組織中不表達(dá)、在一些腫瘤及增殖性病變細(xì)胞中有高表達(dá)。Caspases蛋白在介導(dǎo)細(xì)胞凋亡中處于重要地位,大多數(shù)的刺激性信號(hào)是經(jīng)由Caspase級(jí)聯(lián)反應(yīng),激活并誘導(dǎo)細(xì)胞凋亡的。Livin與其中多個(gè)成員相互作用,抑制細(xì)胞凋亡的執(zhí)行,例如,Livin可結(jié)合caspase-9的前體蛋白及其激活的形式,從而抑制其功能。 Smac/DIABLO是一個(gè)促進(jìn)凋亡的重要相關(guān)蛋白,具有239-氨基酸殘基,一般定位于線粒體的膜間隙。當(dāng)?shù)蛲鲂盘?hào)刺激Smac/DIABLO時(shí),Smac會(huì)伴隨著細(xì)胞色素c的釋放進(jìn)入胞漿,成熟的Smac在離開線粒體后,可與XIAP、Livin等IAPs家族成員鋅指結(jié)構(gòu)域BIR3上的表面溝結(jié)合,而這個(gè)溝是用于結(jié)合caspase9的部位,因而Smac能競爭性去除IAPs對(duì)凋亡的抑制,從而進(jìn)一步激活Caspase級(jí)聯(lián)反應(yīng),最終誘導(dǎo)細(xì)胞凋亡。 Mcm3,即微小染色體維持蛋白3,作為Mcms家族中重要的一員,與生物體DNA復(fù)制過程密切相關(guān)。MCMs家族目前有7種。微小染色體是指酵母中一系列核小體被包裝后形成的結(jié)構(gòu),是酵母復(fù)制的結(jié)構(gòu)基礎(chǔ)。 同經(jīng)典的增殖性標(biāo)記物Ki-67相比,兩者雖都定位于增殖細(xì)胞中。只有Mcm3能夠在相當(dāng)數(shù)量的處于生長靜止期的細(xì)胞中表達(dá)陽性,而Ki-67無表達(dá),可以認(rèn)為Mcm3作為一種細(xì)胞增殖標(biāo)記物,較Ki-67更有意義。 目前關(guān)于Livin, Smac和Mcm3在NP中表達(dá)的相關(guān)研究很少。三者聯(lián)合檢測分析,并與NP臨床分型分期和術(shù)后復(fù)發(fā)率之間做相關(guān)性研究,指導(dǎo)耳鼻喉科醫(yī)師制定更加適宜的個(gè)體化手術(shù)治療及隨訪方案,在國內(nèi)外目前的檢索文件中尚未見報(bào)道。 本文主要研究目的在于：(1)通過聯(lián)合檢測3種典型的細(xì)胞凋亡／增殖相關(guān)因子：Livin, Smac和Mcm3在NP的表達(dá)特點(diǎn),進(jìn)一步探索NP在細(xì)胞凋亡途徑可能的發(fā)病機(jī)制,為進(jìn)一步闡明細(xì)胞凋亡機(jī)制在NP的發(fā)生、發(fā)展中的重要作用,具有較強(qiáng)的理論意義。(2)并通過對(duì)上述3種細(xì)胞凋亡／增殖因子的表達(dá)水平與NP臨床分型分期及術(shù)后復(fù)發(fā)率進(jìn)行相關(guān)性分析,探索一條能較為客觀科學(xué)地預(yù)測NP患者術(shù)后復(fù)發(fā)可能性的分子水平途徑,可作為臨床參考指標(biāo),與鼻科醫(yī)師的臨床經(jīng)驗(yàn)性判斷互為補(bǔ)充,用于指導(dǎo)耳鼻喉科醫(yī)生對(duì)NP患者在手術(shù)治療、圍手術(shù)期處理以及隨訪等方面進(jìn)行個(gè)體化的制定,以改善目前主要憑醫(yī)師臨床經(jīng)驗(yàn)做出判斷的現(xiàn)狀,有效減少復(fù)發(fā),具備實(shí)際的臨床應(yīng)用價(jià)值。 方法：于山東大學(xué)第二醫(yī)院病理科檢驗(yàn)并保存的2005年6月～2009年6月期間入院的單純鼻息肉患者蠟塊中隨機(jī)選取80例,男性52例,女性28例,平均年齡34.5歲(19-57歲),臨床分型分期為Ⅱ型Ⅰ期24例,Ⅱ型Ⅱ期26例,Ⅱ型Ⅲ期16例,Ⅲ型14例。均病理確診為鼻息肉.通過詳細(xì)詢問病史、臨床癥狀、鼻內(nèi)窺鏡檢查、變應(yīng)原皮膚試驗(yàn)(參照2008年WHO制定的《過敏性鼻炎的處理及其對(duì)哮喘的影響》以及中華醫(yī)學(xué)會(huì)呼吸病學(xué)分會(huì)哮喘學(xué)組制定的2008年《支氣管哮喘防治指南》),均排除變應(yīng)性鼻炎、哮喘等變態(tài)反應(yīng)性疾病史。80例患者隨訪復(fù)查期均超過12個(gè)月。隨訪期內(nèi)經(jīng)病理證實(shí)復(fù)發(fā)者(所有復(fù)發(fā)患者均首先在鼻內(nèi)窺鏡復(fù)查時(shí)發(fā)現(xiàn)息肉樣新生物,向患者講明目前病情并由本人簽署我院特殊檢查治療知情同意書后,然后行內(nèi)窺鏡下鼻息肉清理并送病理科再次診斷),Ⅱ型Ⅰ期2例,Ⅱ型Ⅱ期6例,Ⅱ型Ⅲ期6例,Ⅲ型8例。 正常對(duì)照組：12例,取自2008年5月～2009年12月間我院外傷性腦脊液鼻漏患者手術(shù)修補(bǔ)過程中,為正常鉤突粘膜,均排除鼻-鼻竇炎及變應(yīng)性鼻炎、哮喘等變態(tài)反應(yīng)性疾病史(排除方法同鼻息肉組,并結(jié)合鼻竇CT)。 通過免疫組化SP方法,檢測標(biāo)本組織中Livin, Smac及Mcm3的表達(dá),以胞漿和/或胞核中出現(xiàn)棕黃色或棕褐色顆粒為陽性信號(hào),應(yīng)用Olympus顯微鏡和Micro-image圖像處理系統(tǒng),每例標(biāo)本選取3張切片,每張切片均采用雙盲法隨機(jī)觀察10個(gè)高倍視野(顯微鏡下×400),用半定量積分法判斷結(jié)果,所有數(shù)據(jù)經(jīng)統(tǒng)計(jì)學(xué)處理。 結(jié)果：80例標(biāo)本中,Livin表達(dá)陽性片為54例,陽性率為66.7%(54/80),主要表達(dá)在息肉組織內(nèi)增生的腺體/腺管上皮細(xì)胞,定位于胞漿；對(duì)照組12例正常粘膜無1例陽性(0/12)。Mcm3表達(dá)陽性片為46例,陽性率為56.7%(46/80),主要表達(dá)在息肉上皮細(xì)胞,定位于胞核。對(duì)照組正常粘膜4例陽性,陽性率為33.3%(4/12),定位于粘膜上皮細(xì)胞的胞核。Smac表達(dá)陽性為42例,陽性率為52.5%(42/80),主要表達(dá)在息肉上皮細(xì)胞,部分腺體上皮亦可見陽性表達(dá),定位于胞漿。對(duì)照組正常粘膜11例陽性,陽性率91.7%(11/12),主要表達(dá)在粘膜上皮細(xì)胞,定位于胞漿。Livin,Smac在鼻息肉與正常粘膜對(duì)照組之間的表達(dá)差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。Mcm3在鼻息肉與正常粘膜對(duì)照組之間的表達(dá)差異不具有統(tǒng)計(jì)學(xué)意義(P0.05)。Livin, Smac表達(dá)強(qiáng)度在鼻息肉不同臨床分型分期之間具有統(tǒng)計(jì)學(xué)上的差異(P0.05),可以認(rèn)為鼻息肉不同臨床分型分期中Livin, Smac表達(dá)強(qiáng)度是不同的。Mcm3表達(dá)強(qiáng)度在鼻息肉不同臨床分型分期之間不具有統(tǒng)計(jì)學(xué)上的差異(P0.05)。Livin、Mcm3聯(lián)合表達(dá)強(qiáng)度與鼻息肉臨床分型分期之間呈正相關(guān)關(guān)系(R's=0.453,P0.05)；而Smac表達(dá)強(qiáng)度與鼻息肉臨床分型分期之間呈負(fù)相關(guān)關(guān)系(R's=-0.429,P0.05)。Livin、Mcm3聯(lián)合表達(dá)強(qiáng)度與鼻息肉術(shù)后復(fù)發(fā)率之間呈直線正相關(guān)關(guān)系(R=0.9569,P0.05)；而Smac表達(dá)強(qiáng)度與鼻息肉術(shù)后復(fù)發(fā)率之間呈直線負(fù)相關(guān)關(guān)系(R=-0.9781,P0.05)。 結(jié)論：(1)Livin過度表達(dá)可能在鼻息肉內(nèi)腺體發(fā)生發(fā)展的過程中具有重要作用。(2)鼻息肉中存在明顯的Smac表達(dá)受阻。(3)Mcm3標(biāo)志的鼻息肉細(xì)胞增殖活性的提高在其發(fā)病機(jī)制中可能處于一個(gè)相對(duì)次要的地位,而凋亡受抑制可能起首要作用。(4)鼻息肉臨床分型分期高低與凋亡受抑程度之間存在正相關(guān)關(guān)系,其分型分期越高,細(xì)胞凋亡可能越弱。(5)Livin, Smac及Mcm3作為在細(xì)胞凋亡/增殖動(dòng)態(tài)平衡中具有不同作用機(jī)理的三個(gè)重要因子,其聯(lián)合檢測結(jié)果可作為有臨床意義的客觀參考指標(biāo),在分子水平上預(yù)測個(gè)體鼻息肉患者術(shù)后復(fù)發(fā)的可能性；與臨床經(jīng)驗(yàn)相結(jié)合,使耳鼻喉科醫(yī)師對(duì)鼻息肉患者可采取更適宜的個(gè)體化手術(shù)方案、圍手術(shù)期處理及隨訪。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R765.25

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1 黎軍和;何文靜;何友兼;;Survivin和Livin在Dukes'B期結(jié)直腸癌中的表達(dá)及其臨床意義[J];癌癥;2007年05期

2 曠興林;促凋亡因子Smac/DIABLO的研究進(jìn)展[J];國外醫(yī)學(xué).臨床生物化學(xué)與檢驗(yàn)學(xué)分冊(cè);2003年04期

3 李曉明;馬秀茹;路秀英;崔麗萍;董文榮;;凋亡抑制蛋白XIAP在喉鱗狀細(xì)胞癌中的表達(dá)及其臨床病理學(xué)意義[J];臨床耳鼻咽喉頭頸外科雜志;2007年21期

4 陳曉;楊春鹿;趙君;李大朋;張林;;非小細(xì)胞肺癌組織Livin和Smac蛋白表達(dá)及其臨床意義的研究[J];中華腫瘤防治雜志;2008年12期

5 童強(qiáng)松,鄭麗端,阮慶蘭,郭筱蘭,湯紹濤,劉春萍;Smac和Caspase-3基因在神經(jīng)母細(xì)胞瘤中的表達(dá)及其臨床意義[J];中國癌癥雜志;2004年01期

6 魯祥石;劉彥龍;王滌;龐達(dá);崔濱濱;;凋亡抑制蛋白Livin和Survivin在乳腺癌中的表達(dá)及意義[J];中國癌癥雜志;2007年07期

7 董震,關(guān)桂梅,常萬龍;鼻息肉組織中細(xì)胞增殖與凋亡相關(guān)基因蛋白的表達(dá)及意義[J];中華耳鼻咽喉科雜志;2000年06期

8 ;附:慢性鼻竇炎鼻息肉臨床分型分期及內(nèi)窺鏡鼻竇手術(shù)療效評(píng)定標(biāo)準(zhǔn)(1997年,�？�)[J];中華耳鼻咽喉科雜志;1998年03期

9 中華醫(yī)學(xué)會(huì)呼吸病學(xué)分會(huì)哮喘學(xué)組;支氣管哮喘防治指南(支氣管哮喘的定義、診斷、治療及教育和管理方案)[J];中華結(jié)核和呼吸雜志;2003年03期

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