【作者】 李春晖；

【导师】 马忠恕；

【作者基本信息】 吉林大学 ， 耳鼻咽喉科学， 2003， 硕士

【摘要】 恶性肿瘤仍是人类难以攻克的难题之一，揭示肿瘤的发病机制将能够有效指导肿瘤的早期诊断及特异治疗，故对肿瘤发病机制的研究成为学者们工作之中心。喉癌占头颈肿瘤的13.9%，仅次于鼻咽癌和鼻腔癌而位居第三位，近年来喉癌发病率有明显的增长趋势。其发生发展一方面可能是癌基因激活、抑癌基因缺失或突变导致肿瘤细胞的过度增殖，另一方面，也可能是细胞凋亡过程受阻，促使肿瘤细胞存活的结果。当肿瘤细胞增殖和/或凋亡的调节因素改变，则会影响肿瘤的生长。几种细胞凋亡抑制因子包括bcl-2家族和IAPs已被分离鉴定。肿瘤特异性凋亡抑制因子Survivin因其独特的特点，在探讨肿瘤发病机制、早期诊断、靶向治疗、预后等方面的作用，正引起全世界的瞩目。Survivin是1997年新发现的凋亡抑制因子，属于IAP家族，但结构独特，仅含有单一的BIR功能区，没有环指结构；作用于各种凋亡通路末端效应子caspase-3和caspase-7而阻断各种刺激（如Fas、caspase、Bax、某些化疗药物）诱导的细胞凋亡过程；在细胞中的表达具有严格的细胞周期依赖性-G2/M期特异性表达，定位于细胞质；组织分布具有明显的细胞选择性-肿瘤特异性，即选择性地表达于胚胎发育组织和大多数肿瘤组织，在正常成人终末分化组织中不表达；具有天然反意义链EPR-1，通过天然反意义转录物的相互作用可以调节细胞凋亡。bcl-2是最早发现的细胞凋亡调控基因，其作用机制可能与Ca2+依赖性核酸内切酶、抑制氧自由基的产生、控制细胞信号传导来延长细胞生存等有关。bcl-2有许多同源体，这些蛋白在体内通过同源、异源二聚体相<WP=47>互作用，而且促凋亡、抑制凋亡蛋白的相互比例决定着细胞对死亡刺激因素的最终敏感性。Bcl-2在多种因素诱导的凋亡抑制中起作用，如血清饥饿、细胞与细胞外间质粘附丧失等。为探讨喉鳞癌的细胞凋亡情况，我们应用免疫组化方法对细胞凋亡相关基因survivin、bcl-2、bax在喉鳞癌中的表达及其临床相关性进行了研究。通过探讨在喉鳞癌、声带息肉组织中凋亡抑制因子survivin的表达及与bcl-2、bax的关系，阐明凋亡相关因子survivin与bcl-2、bax的异常表达及其相互关系所引起的凋亡抑制在喉鳞癌的病因学中的意义。研究应用免疫组化SABC方法检测54例喉鳞癌手术切除标本石蜡切片组织中survivin、bcl-2、bax的表达情况，并以10例声带息肉组织为对照。结果发现：1、survivin在声带息肉中不表达，在70.4%喉鳞癌中表达(p<0.05)；2、survivin阳性表达率与喉癌患者的年龄、性别、肿瘤大小、临床分型无相关性(p＞0.05)，与喉癌临床分期、组织学分级、淋巴结转移正相关(p<0.05)；3、bcl-2在声带息肉中不表达，在75.9%喉鳞癌中表达(p<0.05)；4、bcl-2阳性表达率与喉癌临床分期、组织学分级正相关(p<0.05)；5、bax在喉鳞癌中和在息肉组织中都表达，Bax在肿瘤组织中表达升高，但两组差异无显著性意义(p＞0.05)。5、survivin阳性表达率与bcl-2的表达正相关(p<0.05)；与bax的表达无相关性(p＞0.05)。研究结果表明，①survivin在终末分化的成人组织中不表达，在喉鳞癌细胞中表达，具有组织表达特异性，参与肿瘤的发生。②Survivin高表达与喉癌的临床进展期和组织学分类中高度恶性及预后不良等有关，提示Survivin参与喉癌的发生发展，与预后密切相关，从而认为检测Survivin为诊断肿瘤、预测肿瘤的发生发展和预后提供一新的指标。③喉癌细胞中，<WP=48>bcl-2表达增高，抑制细胞凋亡，推测因此增多细胞内其它基因发生变异的机会、逃脱机体正常的监视并清除异常细胞的作用而达永生化，是喉癌发生的原因之一。④bcl-2的高表达与喉癌临床分期、组织学分级相关，说明Bcl-2表达改变影响肿瘤恶性行为。⑤Bax在正常组织中发挥促进凋亡功能，而在肿瘤组织中表达升高，提示喉癌细胞中存在非肿瘤细胞中所存在的凋亡促进机制，而且其作用高于非肿瘤组织。本实验的研究结果进一步证实了喉鳞癌同时具有抑制凋亡与促进凋亡两种机制，但抑制凋亡作用远超过促进凋亡作用导致癌细胞增殖超过凋亡，提示细胞凋亡抑制所致细胞永生化在肿瘤发生发展和预后都起着重要作用；在肿瘤的发生发展中，两种凋亡抑制因子survivin和bcl-2既执行各自不同的抗凋亡功能，又协同发挥抗凋亡效应引起凋亡调节紊乱，有助于细胞的恶性转化和肿瘤的进展，为进一步揭示肿瘤的发病机理提供了实验室依据和理论基础。另一方面，对survivin和bcl-2进一步研究可以指导肿瘤的治疗：①由于靶向Survivin抗肿瘤治疗能促进肿瘤细胞的凋亡并抑制其增殖，正常组织不受影响而免于受损，具有较好的特异性及安全性；②靶向Survivin抗肿瘤治疗可以克服反义寡核苷酸基因治疗存在的特异性差，基因传递不完全的缺点；③Survivin表达的抑制还可以对抗肿瘤的耐药性、提高肿瘤细胞对许多化疗药物的敏感性；④Survivin靶向治疗可与bcl-2的靶向治疗、抗癌药物联合发挥协同作用。总之，对Survivin、bcl-2等深入研究必将为探讨肿瘤的发生机制提供新契机；检测Survivin为诊断肿瘤、预测肿瘤的发展和预后提供一新的指标；从而为指导肿瘤治疗开辟新天地。更多还原

【Abstract】 Tumor is still one of unsolvable problems. To reveal pathogenesy of tumor can help to direct early tumor diagnosis and specific therapy. So research of tumor pathogenesy has become more and more important. The morbidity of Laryngocarcinoma is second only to nasopharyngeal carcinoma and nasal cavity carcinoma. Recently, this morbidity has been evidently increasing. Although much research had been done, pathogenesy of tumor has been uncertain. Laryngocarcinoma formation is a process containing a few oncogenes , anti-oncogenes and multiple stages. On the one hand, oncogenes activation, anti-oncogenes deletion or mutation lead to tumor cell excess proliferation; on the other hand, it may be the result that inhibitors of apoptosis aberrantly prolongs tumor cell viability . There is a delicate balance between cell proliferation and apoptotic, which are two important factors in maintenance of cell and tissue homeostasis. If the regulation factors change, tumor formation and growth should be effected. So far, several apoptosis inhibitors such as bcl-2 family and inhibitors of apoptosis family (IAPs),have been identificated.IAP proteins were discovered in baculoviruses and are typically characterized by 2-3 baculovirus IAP repeats (BIR) and a RING finger motif. These proteins suppress apoptosis through direct caspase and pro-caspase inhibition, (primarily caspase 3 and 7). Recently, the cancer field has become greatly excited by the discovery of Survivin, one of the IAPs, that functions in vertebrates to regulate both cell proliferation and apoptosis. Survivin contains a single BIR and lacks a carboxyl-terminal RING finger. Although undetectable in terminally differentiated adult tissues, survivin is present <WP=50>during fetal development and in most transformed cell lines and cancers tested to date. Even more importantly, survivin expression appears to be homogenous within the tumor environment, a feature unusual in a disease characterized by spontaneous mutation. Expression of survivin was observed in the cytoplasm, and at specifical stage of the cell cycle-G2/M. Furthermore, survivin has a natural antisense strand, effector cell protease receptor-1(EPR-1),so regulation of transcriptions of these two complementations can modulate apoptosis.bcl-2 is the firstly found apoptosis regulation gene, and its mechanism of action may be concerned with a few factors such as Ca2+ dependent endonuclease, anti-oxyradical, control of cell signaling and so on. bcl-2 has several homologizations, and these proteins interact to promote or inhibite apoptosis. Bcl-2 play a part in apoptosis inhibition induced by many factors.To identify the role of apoptosis inhibition induced by overexpression of survivin and its relationship to bcl-2 and bax in Laryngocarcinoma pathogenesis, immunohistochemical staining of survivin,bcl-2 and bax by using the monoclonal or multiclonnal antibody were performed by the standard streptavidin-peroxidase (SP) technique for the 64 paraffin sections which included 10 polyp of cord as control, and 54 Laryngocarcinoma. Overexpression of survivin(70.4%), bcl-2(75.9%) and bax(61.1%) were observed in Laryngocarcinoma. No immunoreactivity of survivin and bcl-2 were seen in the polyp of cord while positive rate of bax is 30.0% . The positive rate of survivin was related to the clinical stage, the histological grade and the cervical lymphatic metastasis of the tumor, and not to patients’ age, sex, tumor size or clinical classification. The positive rate of bcl-2 was related to the clinical stage, the histological grade and the cervical lymphatic metastasis of the tumor. Moreover, there was the correlation between survivin and bcl-2, and not between survivin and bax.Our studies demonstrated that the action of apoptotic inhibition of survivin may participate in the onset and progression of laryngeal carcinoma. Survivin could become a new diagnostic , prognostic and therapeutic marker in <WP=51>laryngeal carcinoma. The overexpression of bcl-2 is the essential event in the early stage of laryngeal carcinoma becau更多还原