【作者】 宫琳琳；

【导师】 邵淑娟；

【作者基本信息】 大连医科大学 ， 人体解剖与组织胚胎学， 2004， 硕士

【摘要】 目的:肺癌目前是世界上发病率和死亡率最高的恶性肿瘤,严重影响人类健康,但其癌变机制仍不很清楚。近年来,随着肿瘤分子生物学的深入研究,发现大多数人体恶性实体肿瘤组织DNA出现癌基因的活化、抑癌基因的失活遗传物质变异。正常细胞的癌变是多因素、多阶段及多基因作用的结果。而抑癌基因一般为隐性基因,要在一对等位基因都丢失或失活后,它们的作用才能被发现。一旦失活,可能使癌基因失去控制,过度表达,导致癌变。因此抑癌基因的失活在多种癌变过程中起着重要的作用。抑癌基因的失活可由等位基因丢失,染色体重排,点突变和异常甲基化等引发。8p的LOH是肺癌发生发展过程中的早期事件,也是肺癌发生过程中的常见的遗传事件,8p的缺失是多种肿瘤发生发展中重要的遗传学改变。人们一直期望如同在3p的缺失区域发现FHIT基因,在9p的缺失中找到p16基因,在17p的缺失中锁定p53基因那样,在8p发现重要的侯选抑癌基因。然而迄今为止在8p中一直没有发现公认的在多种肿瘤中发挥作用的抑癌基因,这和8p在多种肿瘤中的高频缺失的现象是不吻合的。我们在对8p22区域的LOH进行分析时注意到Fez1(F37/Esophageal cancer-related gene-coding leucine-zipper motif,F37/食管癌相关基因编码亮氨酸拉链基因序列,简称FEZ1/LZTS1)基因——一个候选的抑癌基因。本实验以临床肺癌组织标本及相应的远端正常肺组织为对象,探讨与候选抑癌基因Fez1紧密连锁的2个微卫星多态位点的缺失率及其在肺癌中的表达及其与临床<WP=5>病理因素的关系。方法:实验分为三部分:(1)选取与候选抑癌基因Fez1紧密连锁的微卫星多态位点D8S261和D8S233,然后对32例肺腺癌组织进行两个位点的杂合性缺失(LOH)分析。(2)对32例肺腺癌组织及其相应的远端正常肺组织利用S-P法进行免疫组化分析。(3)选取另外10例肺癌组织标本及相应的正常肺组织,用Western blot检测Fez1表达的差异。结果:对候选抑癌基因Fez1紧密连锁的两个位点D8S261和D8S233进行LOH分析,结果表明:两个位点D8S261和D8S233的杂合率分别为64.52%(20/31)和46.88%(5/32),LOH阳性率分别为60.0%(11/18)和61.0%(9/15)。免疫组化分析显示Fez1蛋白在正常的支气管上皮中100%表达,在27例有效检测病例中,肺腺癌阳性表达率为33.33%,下调表达的病例占66.67%。Western blot 检测结果显示在肿瘤组织中Fez1表达明显低于正常组织。结论:1.32例肺腺癌的8p微卫星多态性标记的LOH频率表明所检测位点中D8S261的LOH发生频率与肺腺癌分期正相关(P<0.05)。2.Fez1在正常肺组织和癌旁支气管上皮细胞中正常表达,在肿瘤组织中的下调表达率达66.67%,其与肺腺癌的病理分级、临床分期无相关性(p>0.05)。在混合性的气管腺中,在浆液性腺泡中正常表达,在黏液性腺泡中不表达。3.Western blot检测观察,结果表明Fez1在肺癌和远端正常组织中的表达存在差异。4.肺腺癌组织中Fez1蛋白的丢失是频发事件,Fez1可能是肺腺癌发生过程中重要的候选抑癌基因,LOH是其失活机制之一,可能还存在LOH以外的其它失活机制。更多还原

【Abstract】 Objective Lung cancer is the most common malignant tumor worldwide, with a high incident and mortality rate. However its carcinomatous mechanism is still unknown. Recently with the lucubrating the Oncology and Molecular Biology, people found that the DNA of the most malignant tumors would be aberrant including the activating of oncogenes and inactivating of tumor suppressor genes. Carcinomatous changing of normal cells result from the accumulation of genetic alterations at specific chromosomal regions involving a multistep process. Tumor suppressor genes are recessive genes. Their functions would not be found until the couple of the alleles lose or inactivate. While inactivating of tumor suppressor genes could make oncogenes express excessively, which would lead to cancer. So inactivating of tumor suppressor genes is a critical event to lung cancer pathogenesis. The main patterns include loss, mutation and methylation. Loss of heterozygosity (LOH) of 8p is an early and frequent event in lung cancers. Allelic losses on 8p have also been reported as an important genetic changing in many kinds of cancers, including prostate, breast, head-and-neck, esophageal and urinary bladder carcinomas. People always hope to find an important candidate tumor suppressor gene on 8p, just like finding FHIT on 3p, p16 on 9p and p53 on 17p. But until now people have <WP=7>not found a legal tumor suppressor gene on 8p which could exert effects in many kinds of tumors, this is not tally with the high frequent loss on 8p. When we studied 8p22 region and took LOH analysis, we noticed Fez1 (F37/Esophageal cancer-related gene-coding leucine-zipper motif) gene---a candidate tumor suppressor gene. We here take adenocarcinomas and accompanying nonmalignant tissues as the objectives for the study. Our goal is to investigate the two microsatellite sites inside or flanking to the Fez1 and its expression in pulmonary adenocarcinoma and their relationship to clinicopathological factors. Methods Our work is composed of three parts: (1) Microsatellite analysis was performed by using the primers (D8S261 and D8S233) that amplified polymorphic sequences inside or flanking to the Fez1 in the tumor tissues collected from 32 cases of primary adenocarcinoma. (2) Immunohistochemical S-P technique was performed to detect the expression of Fez1 protein in the tumor tissues and accompanying nonmalignant tissues. (3) Utilize Western blot to check the different expression of Fez1 in another 10 tumor tissues and accompanying nonmalignant tissues.Results For loci D8S261 and D8S233, LOH was found in informative cases was 60.0% (11/18) and 61.0% (9/15), respectively. Fez1 protein was 100% detectable in normal bronchial epithelium. The Fez1 protein was detectable in 33.33% of the samples, expression of Fez1 protein was down regulated in tumor tissue is 66.67%, the other 5 cases was not done. No correlation was found between the Fez1 protein expression and its clinicopathological parameters such as pathological group, clinical staging (p>0.05). The results of the Western blot show that the expression of the Fez1 in tumor tissues is obviously lower than normal tissues.Conclusion1. The result of Microsatellite analysis showed that a significant <WP=8>correlation between LOH of D8S261 and tumor grade(P<0.05).2. Scontaining protions of normal bronchial epithelium were uniformly positive in all cases. Overall in 27 informative cases, 66.67% (18 cases) primary tumors with reduced or absent Fez1 expression, compared with normal bronchial epithelium. In mixed tracheal glands, Fez1 express normally in serous alveoli and on expression in viscous alveoli. 3. The result of Western blot showed that the expressions of Fez1 in lung cancer and accompanying nonmalignant tissues were different.4. Loss of Fez1 expression is a frequent event in pulmonary adenocarcinoma and it may be one of the important candidate tumor suppressor gene in carcinogenesis of pulmonary adenocarcinoma. Its inactivation is attributable to several factors not only LOH of Fez1 gene but更多还原