【作者】 姜润德；

【导师】 李春海；

【作者基本信息】 中国人民解放军军事医学科学院 ， 医学免疫学， 2003， 博士

【摘要】 鼻咽癌是中国南部及东南亚地区最常见的恶性肿瘤之一，该地区鼻咽癌的发病率最高可达50／10万，死亡率占全部恶性肿瘤的2.81％。与其它恶性肿瘤相比，它具有以下几个方面的显著特点：(1)具有非常明确的地理分布；(2)与EB病毒的感染有特殊的相关关系；(3)绝大多数鼻咽癌属未分化或低分化癌，恶性程度高，浸润生长快，极易发生癌细胞的远处转移和局部治疗的不彻底。在临床上化疗是鼻咽癌最主要的辅助治疗手段，然而癌细胞耐药性的产生常常引起药物治疗的失败。因此体外建立鼻咽癌耐药细胞系，筛查鼻咽癌耐药相关基因，从基因组DNA水平和基因表达水平寻找在耐药产生中具有关键作用的分子改变，对于全面了解肿瘤细胞的耐药机制，寻找逆转药物耐受的分子靶点具有重要的理论和现实意义。 肿瘤细胞耐药产生的机制从化疗用于肿瘤的治疗之初便开始受到了广泛的研究。人们很早就认识到肿瘤细胞具有多药耐药现象，肿瘤的多药耐药性(Multidrug Resistance,MDR)指的是肿瘤细胞在接触某一种化疗药物后，不仅对此种药物产生耐受性，而且对其它的结构和功能不同的多种药物也产生交叉耐受性。多药耐药是恶性肿瘤难治和复发的重要原因之一，客观地讲，其本质是肿瘤细胞在生存压力之下建立起来的一种适应过程。肿瘤细胞可以通过多种机制产生MDR，其中细胞膜上表达药物排流泵、通过泵机制把药物从细胞内排出细胞外是其最主要的耐药方式。其它尚包括细胞解毒能力的增强、DNA修复能力的增强、药物靶蛋白如拓扑异构酶量或活性的改变、蛋白激酶C各种同工酶活性和表达水平的改变等。然而迄今以这些分子作为靶点的耐药逆转研究仍存在着许多难以克服的问题。肿瘤耐药是否还存在其它的分子机制?另外，由于鼻咽癌发生的特殊性和其独特的生物学特点，鼻咽癌细胞耐药的产生是否也具有特殊性? 本研究首先用鼻咽癌药物敏感细胞CNE-2作为亲本细胞诱导建立耐药细胞系。在此基础上，用改良的消减杂交方法筛选鼻咽癌耐药相关基因，用比较基因组杂交技术研究耐药细胞在基因组DNA水平上可能存在的异常扩增和缺失，以此探讨与鼻咽癌耐药性产生有关的分子机制。博士学位论文:鼻咽癌耐药细胞系(CNE一2/CDDP)的建立及耐药相关基因的筛选研究主要包括三个部分的内容:一人鼻咽癌顺铂耐药细胞系(CNE一2/C DDP)的建立 以鼻咽癌低分化细胞系CNE一2为研究对象，以鼻咽癌一线化疗药物顺铂 (CDDP)为诱导剂，采用大剂量冲击与剂量逐渐递加相结合的方法，诱导建立人鼻咽癌耐药细胞系CNE一2/CDDP。耐药指标的检测包括MTT法测定耐药细胞和其亲本细胞对不同药物的敏感性，流式细胞术测定细胞内荧光药物罗丹明的蓄积，细胞生长曲线和倍增时间的测定及细胞的形态学观察。结果:所建立的人鼻咽癌耐药细胞系CNE一2/CDDP对CDDP的耐药指数为27.9，对5一氟尿嗜睫(5一FU)及长春新碱(VCR)的耐药指数分别可达227.9和55.5，表明其具有多药耐药的特性。流式细胞术测定显示细胞被荧光药物罗丹明作用后，CNE一2/CDDP的细胞内罗丹明荧光强度显著低于CNE一细胞(1 2.98/243.62)，说明耐药细胞能有效地把细胞内药物排出细胞外。CNE一2/CDDP的细胞倍增时间也明显延长 (26h/19h)。在形态学上，CNE一2/CDDP细胞较CNE一2细胞表现为体积减少，形态变圆，在培养瓶中易形成克隆集聚现象。 结论:本研究建立了稳定的人鼻咽癌耐药细胞系CNE一2/CDDP，为进一步研究恶性肿瘤耐药性产生的机理建立了理想的耐药细胞模型。二.用改良的消减杂交方法克隆鼻咽癌耐药相关基因 在建立鼻咽癌耐药细胞系的基础上，采用基于PCR技术的改良的消减杂交方法筛选并克隆鼻咽癌耐药相关基因。对获得的系列差异表达片段连接T一easy载体，转化入JM109菌株，建立消减文库。并对差异表达的基因片段进行一系列鉴定:质粒的酶切鉴定、反向点杂交鉴定、RT一PCR鉴定和Northern b10t鉴定。对经过鉴定的基因进一步做DNA序列测定并利用NCBI的GenBank数据库(http://w ww. nCbi·nlm.nih.gov)对其进行比较分析，以初步了解差异表达基因的性质并分析其在鼻咽癌耐药性产生中可能的作用机理。结果共发现了6个在耐药细胞中差异表达的基因片段，其中3个为耐药细胞所高表达，3个在耐药细胞中表达下调。高表达的序列中有一个是功能未知的恶性肿瘤相关基因，有完整的开放读框，其编码产物是含有79个氨基酸的DC13蛋白。其余两个序列分别与泛素C基因和线粒体编码基因NADH脱氢酶亚单位2(ND2)有同源性。在耐药细胞中表达受到抑制的基因序列分别与细胞色素氧化酶亚单位1(COXI)、核糖体蛋白大亚基27单体(RPL27)以及核糖体蛋白小亚基27单体(RPS27)基因有同源性。博士学位论文:鼻咽癌耐药细胞系(CNE一2/CDDP)的建立及耐药相关基因的筛选 由于改良的消减杂交法所克隆的基因片段从理论上讲应该是表达产物mRNA的全长，因此，所克隆的cDNA如果有完整的开放读框(ORF)，该序列就有可能包含完整的cDNA。本研究所克隆的6个基因片段经分析其中5个有完整的ORF，推测有可能代表不同的基因表达产物。为此对该更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in south China and southeast Asian countries. The highest incidence of NPC in this district may reach 50/100, 000, and its mortality rate is 2. 81% among all the malignancies. In comparing to other malignant tumors, NPC has its unique properties, which are: (1) unique geographical distribution; (2) specific relationship with EB virus; (3) most cases of NPC belong to non or low differentiate type. With its notorious properties of very low differentiation and rapid invasive growth, most cases are inclined to distant metastasis and not ready to be completely cured by local therapies, so chemotherapy constitutes one of the chief methods in our treatment of this disease. However, the appearance of drug resistance often causes failure of chemotherapy. For overcoming drug resistance, it is of great importance to establish a drug-resistant cell line in vitro and to screen drug-resistant associated genes. To search for the key molecular alteration in genomic DNA level and in gene expression level, and to identify potential molecular targets should be of great significance in our understanding of drug-resistance mechanism and in our struggle against this cancer.The mechanisms of drug-resistance have been videly investigated ever since the application of chemotherapy in the treatment of cancers. The multidrug resistance (MDR) is defined that the cancer cells may establish the ability to resist different drugs whose structure and pharmacodynamics are not alike, after they are exposed to only one drug. MDR is one of the most common causes of chemotherapy failure. However,from a reasonable view, its essence is an adaptive process of tumor cells under an unfavorable survival circumstance. Many mechanisms might contribute to the appearance of MDR, such as the expression of pump protein, the P-glycoprotein in the membrane, the enhanced detoxifcation ability, the enhanced repair ability of damaged DNA, the quantity or activity alteration of drug target proteins such as topoisomerases and the alteration of activity and expression of protein kinase C. However, many obstacles still confront us in our struggle against drug resistance when we try to use these molecules as potental targets. Are there any other molecular mechanisms of drug resistance? Furthermore, considering the unique characteristics of NPC, are there any other unique mechanisms of drug resistance in NPC?This study was designed to establish a drug-resistant cell line from a human nasopharyngeal carcinoma cell line CNE-2, and to screen human nasopharyngeal carcinoma drug-resistant genes by combining a new strategy based on improved subtractive hybridization with the technique of comparative genomic hybridization.This project includes three parts:1. Establishment of a Human Nasopharyngeal Carcinoma Drug-Resistant Cell Line CNE-2/CDDPThe drug-resistant cell line was established by a program of treating the human nasopharyngeal carcinoma cells CNE-2 in the medium with repeated sharp high and then low but gradually increasing concentration of cisplatin. Drug sensitivity was measured by MTT assay of CNE2/CDDP and its parent cells to different drugs. Fluorescence activated cell analysis (FACS) was employed for determining the concentration of fluorescence dye rhodamine 123 within the cells. Also measured and observed including cell growth curve, doubling time and cell morphology. It was shown that the resistance indexes of CNE-2/CDDP to cisplatin (cDDP), fluorouracil (5-FU) and vincristine (VCR) were 27.9, 227.9 and 55.5 respectively, indicating its multi-drug resistant property. FACS analysis showed that the concentration of rhodamine 123 was much lowerin CNE-2/CDDP cells than that of CNE-2 cells (12.98 vs. 243.62). The CNE-2/CDDP cells appear smaller, more regularly round, with longer doubling time (26h vs. 19h) than CNE-2 cells.From the above bioloical characters we conclude that a stable cell line of multi-drug resistance, which is defined CNE2/CDDP was established. It更多还原