【作者】 贺光；

【导师】 孙开来；

【作者基本信息】 中国医科大学 ， 遗传学， 2002， 博士

【摘要】 1971年Knudson提出的二次突变假说是一个为肿瘤遗传学研究学者所普遍接受的关于肿瘤发生机理的科学预言，指导了二十几年来肿瘤发生分子机制的研究。目前，与肿瘤发生相关的抑癌基因有20余种，癌基因已逾100种。肿瘤的发生是一个多基因参与的、多阶段的复杂过程；不同种类的肿瘤在不同的发展阶段，涉及的肿瘤癌基因和抑癌基因的种类也不一样。因此，探讨不同类型肿瘤相关癌基因和抑癌基因是认识肿瘤发生分子机理、开展基因诊断和基因治疗的基本前提。包括肿瘤在内的疾病致病基因的克隆和定位是人类基因组计划的一项重要内容。所以，我国根据自己的遗传学发展状况和病源优势，紧紧围绕基因组计划，积极开展致病基因的克隆和定位工作。其中，肿瘤相关基因的克隆、定位是肿瘤分子遗传学研究的热点之一。 喉癌是上呼吸道常见的恶性肿瘤，占头颈部癌的绝大部分，约占人体恶性肿瘤的1％～8.4％，病理分型93％～99％为喉鳞状细胞癌。临床资料表明，我国东北地区的发病率呈逐年上升的趋势，可能与吸烟、饮酒及寒冷等环境因素有关。虽然手术、放疗、化疗可以在不同程度上缓解病人的痛苦，但是，治疗后常造成功能障碍、局部畸形，而且复发率较高，严重威胁患者的身心健康。目前，喉癌分子遗传学方面的研究资料较少，虽然有ras、c-myc、EFGR、PRAD、Int-2等癌基因和p53、Rb、p16、FHIT等抑癌基因与喉癌的相关性研究，但是，尚无明确的喉癌相关基因克隆的报道。因此，充分利用这一病源优势开展喉癌相关基因的克隆及鉴定研究是从我国国情出发，实施人类基因组计划的一个重要策略，将填补此领域研究的空白。 本文采用cDNA-代表性差异分析(cDNA-Representational Difference Analysis，cDNA-RDA)方法发现并鉴定喉鳞状细胞癌(laryngeal squamous cell carcinoma，LSCC，以下简称喉癌)新的相关基因，将为喉癌的基因诊断和基因治疗的研究提供靶基因。 材料和方法 组织标本：喉鳞状细胞癌标本来源于中国医科大学附属第一临床学院和沈阳市空军463医院耳鼻喉科患者，手术切取喉癌组织及距癌组织1厘米以上的癌旁正常组织。手术切取喉癌组织及癌旁组织均经病理证实。以我室自建方法进行手工显微切割取材，病理证实瘤细胞超过80％。所有喉癌病例均按1987年UICC喉癌分期标准进行诊断，所有患者术前均未接受放疗和化疗。 1．总RNA提取及mRNA的分离纯化 采用 TRIZOI试剂（IBCO BR）分别提取喉癌及癌旁组织中的总 RNA；用 Poly AT Tract mRNA Isolation System试剂盒（Promega）分离 mRNA。 2．CDNA双链合成 用 Promeg。公司 Universal RiboClone cDNASynthesis System试剂盒分别合成喉癌及癌旁组织 CDNA双链。 3．cDNA－RDA 用Mbo（Promega）限制性内切酶，分别消化喉癌及癌旁组织的cDNA，再由 W DNA连接酶催化连接接头R－24八，然后进行PCR扩增，纯化去除接头并换接头J－24／12，Tester与 Driver经 Gel Works Advance软件定量后按1：10O比例在＊E B uffeuffer中进行第一轮消减杂交，巩R扩增产物即Dpl。同上述方法再进行第二、三轮消减杂交O：400；l：80000X接头分别为 N－24／12、J－24／二二，分别得到N、Dp3。 4．转化和测序 将… 的四个扩增片段分别与pGzuT载体连接，转化后提取阳性克隆质粒DNA，PCR扩增和限制性内切酶酶切鉴定后用 Biy Dye试剂盒（PE）及引物末端标记法分别对插人片段进行自动及手工测序，并在国际互联网上进行同源性比较和分析。 5．Northern blot 采用随机引物法以 a－’中标记DP3的四条表达差异片段河st和Nco酶切后）作为探针（GAPDH作为内参照人取配对癌旁组织及癌组织总RNA25 ug进行甲醛变性琼脂糖凝胶电泳，转膜、预杂交、杂交、洗膜，－70℃放射 ·2·自显影。 6．CK13基因和TGM3基因的LO H分析 选择CK13基因和TGM3基因内部和两端的微卫星多态标记引物，以同位素掺人法进行 PCR扩增，30个循环，条件分别为 94t 30－505，55－61T 30－605，72T45－605，6％变性聚丙烯酸胺凝胶电泳，－70℃放射自显影。与癌旁组织相比，癌组织DNA的扩增条带消失或相对密度减少50％以上判定为LOH，所有阳性及可疑病例均重复实验。 7．CK13蛋白的免疫组化及Western blot研究 选用抗CK13单克隆抗体，应用迈新公司的SP法免疫组化试剂盒，进行癌旁正常组织和癌组织的免疫组织化学染色。Western blot实验中共检测 18对上述标本，配对肿瘤和正常组织加人裂解液门 TisK，140mM NaCI，smM EDTA，二邮 Titon X－100，smM D’IT，pH 7．6），匀浆及超声充分裂解，沉淀重悬于高盐溶液，以 TBS洗涤后机械性溶解于 8 M尿素，12．5％SDSPAGE，转膜后一抗孵育，PBS洗三次，H抗（偶联碱性磷酸酶）孵育后催化CSPD荧光底物发光，室温放射自显影。 8．TGM3基因的 RTICR检测 应用 Primer 3软件进行引物设计，选择 p吧ctin作为内对照更多还原

【Abstract】 IntroductionTwo hit hypothesis (Knudson, 1971) is a scientific prophecy about the mechanism of tumorigenesis which is generally accepted by cancer geneticists, and has directed the study of molecular mechanism of tumorigenesis for morethan 20 years. Mutation refers to those of tumor related genes-oncogenes andtumor suppressor genes (TSG). Till now, more than 100 oncogenes and 20 TSGs have been found. In different developmental stages, different oncogenes and TSGs are involved in various tumors. Tumorigenesis is a complex multi-stages process in which multi-genes are involved. Therefore, the detection of oncogenes and TSGs in different tumors is the basic prerequisite to understand the mechanism of tumorigenesis and to conduct gene diagnosis and gene therapy. Cloning and location of pathogenetic genes including tumor and other diseases are one of the major contents in human genome project. Because of the advantage of patients’ resources in our country, cloning and location of pathogenetic genes according to our developmental status of genetics are of hot spots.As the majority of head and neck cancer, laryngeal carcinoma, which is common in upper respiratory tract, accounts for 1%-8. 4% of human malignant tumors. Clinical data showed a tendency of increasing incidence in Northeast part of China these years, which may relate to smoking, drinking and cold weather. Although the patients’ pain can be released in some degree by surgery, radiotherapy and chemotherapy, the malfunction and local deformity after treatment and the high recurrent rate threaten the health of patients physically and mentally. There are few data about molecular genetics study in laryngeal cancer at present. Although some authors reported that laryngeal cancer was related to oncogenes ( ras, c-myc, EGFR, PRAD, Int-2, etc ) and TSGs ( p53, Rb,p 16, etc) , no clear report about gene cloning related to laryngeal carcinoma was reported. It is important for us to clone and locate laryngeal cancer-related genes by taking full advantage of patients’resources in our country and participating in human genome project so as to fill in the gaps in this field.In our study, we used cDNA-Representational Difference Analysis method to look for and identify novel genes related to laryngeal squamous cell carcinoma, and it would provide target genes for gene diagnosis and gene therapy.Materials and methodsSamplesMatched normal and tumorous tissues from patients who suffered from laryngeal carcinoma were pathologically confirmed. Manual microdissection confirmed that the cancerous cell accounted for more than 80% of total cells. cDNA-Representational Difference Analysis, Cloning and SequencingTotal RNAs were extracted from the samples mentioned above by TRIzol reagent ( GIBCO BRL Co. ) , then mRNAs were isolated by mRNA purification kit (Promega). Double strands cDNA were synthesed, then cutted by Mbo I (Promega) , ligated to R-Bgl-12/24 adaptor after purification. Oligonucleotides were annealed to each other, then incubated for 12 h at 12 - 16 C. Multiple PCR reactions were set up to generate the initial representations after dilution. The R-adaptors ( derived from cancerous tissues) were removed from the representations with same endonuclease, and the digest was phenol extracted and ethol precipitated to form driver, and the tester ( derived from normal tissues) was ligated to the J-Bgl-12/24 adaptor in the manner described above. The first subtractive hybridization was carried out at the ratio of 1: 100, in 41 EPPS buffer at 61 C for 20 h, then the diluted hybridization mix was amplified, 18 cycles (3 min, 95 C;1 min 72 C) ,the 24 bp adaptors acted as primers, then got the first differential products (Dp1). The latter two rounds of subtractive hybridization were performed at the ratio of 1:400 and 1 : 80000 respectively. Dp3 was composed of four fragments and they were purified and cloned into pGEM-T vec-tor. The four clones were then sequenced by ABI 377 Genetic analyser or artificial sequencing after confirmed by c更多还原