人鼻咽癌顺铂耐药细胞系(CNE2/DDP)的建立及耐药相关基因的筛选

背景与目的：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导致药物治疗的失败,因此,筛查鼻咽癌耐药相关基因,寻找逆转药物耐受的分子靶点具有重要的现实意义。本研究首先用鼻咽癌药物敏感细胞CNE2作为亲本细胞诱导建立耐药细胞系,并在此基础上,筛选鼻咽癌耐药相关基因,探讨耐药性产生的分子机制。方法：以顺铂（cisplatin,DDP)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CNE2／DDP。采用MTT法测定药物的敏感性,流式细胞术测定细胞周期及细胞内荧光物罗丹明的蓄积,并进行细胞生长曲线测绘,倍增时间测定及细胞形态学观察。随后,采用基于PCR技术改良的消减杂交法筛选并耐药相关基因,反向点杂交鉴定排除假阳性,DNA测序分析差异表达片段,RT－PCR对差异表达的基因片段做进一步验证。结果：所建立的人鼻咽癌耐药细胞系CNE2／DDP对DDP的耐药指数为27．9,对5－氟尿嘧啶（5－fluorouracil,5-FU)及长春新碱（vincristine,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征,流式细胞测定显示耐药细胞内罗丹明的蓄积明显低于敏感细胞（12．98vs,243.62),镜下观察耐药细胞体积变小,形态变圆,细胞倍增时间明显延长（26hvs,19h)。消减杂交发现了6个差异表达的基因片段,其序列与人已知基因均有部分同源性,其中3个在耐药细胞中高表达,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,有完整的开放读码框,其编码产物是含有79个氨基酸的DC13蛋白,其余两个序列分别是泛素C基因和线粒体编码基因NADH脱氢酶亚单位2（NADH dehydrogenase subunit 2,ND2)。表达被抑制的基因序列分别是细胞色素氧化酶亚单位1（cytochrome C oxidase subunit 1,COX1),核糖体蛋白大亚基27单体（ribosomal protein L27,RPL27)以及核体蛋白小亚基27单体（ribosomal protein S27,RPS28).结论：建立了稳定的人鼻咽癌耐药细胞系CNE2／DDP,为进一步研究恶性肿瘤的耐药机理建立了理想的耐药细胞模型,改良的消减杂交方法是克隆差异表达基因的有效方法,有多个功能已知和未知的基因如DC13,COX1及核糖核蛋白体基因等可能共同参与了鼻咽癌耐药性的产生。

Establishment of a human nasopharyngeal carcinoma drug-resistant cell line CNE2/DDP and screening of drug-resistant genes

BACKGROUND & OBJECTIVE: Chemotherapy constitutes one of the chief supplementary methods in the treatment of nasopharyngeal carcinoma (NPC). However, the appearance of drug resistance often causes failure of chemotherapy. For overcoming drug resistance, it is of great importance to screen drug-resistant associated genes so as to identify potential molecular targets. This study was designed to establish a drug-resistant cell line from a human nasopharyngeal carcinoma cell line CNE2, and to screen human nasopharyngeal carcinoma drug-resistant genes by a new strategy based on improved subtractive hybridization. METHODS: The drug-resistant cell line was established by a program of treating the human nasopharyngeal carcinoma cells CNE2 in the medium with repeated sharp high and then low but gradually increasing concentration of cisplatin. Drug sensitivity was measured by MTT assay. Fluorescence activated cell analysis(FACS) was employed for determining the concentration of fluorescence dye rhodamine 123 within the cells. Cell growth curve, doubling time, and cell morphology were measured and observed. The drug-resistant genes were screened by a new strategy of PCR-based subtractive hybridization.Sequencing and blast analysis were performed after the differentially expressed genes had been verified by reverse dot blotting. The result was further confirmed by RT-PCR. RESULTS: The resistance indexes of CNE2/DDP to cisplatin (DDP), 5-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 lower in CNE2/DDP cells than in CNE2 cells (12.98 vs. 243.62). The CNE2/DDP cells appeared smaller, more regularly round, and longer doubling time (26 hours vs. 19 hours) than CNE2 cells. Six differentially expressed sequences were discovered using improved subtractive hybridization; all of them were found to be homologous to known genes after sequencing analysis. Three of them were highly expressed in CNE2/DDP cells. Among them, one sequence, which encodes a 79 amino acid protein,known as DC13 protein (DC13), was a function unknown gene which has certain relationship with malignancy. The other two sequences were ubiquitin C gene and NADH dehydrogenase subunit 2 (ND2) gene, respectively. The other three of the six sequences, whose expression were inhibited in CNE2/DDP cells, were cytochrome C oxidase subunit I(COX1), ribosomal protein L27(RPL27),and ribosomal protein S27 (RPS27) genes, respectively. CONCLUSION: A drug- resistant cell line CNE2/DDP, which showed a typical resistant phenotype to anti-cancer drugs was established. The PCR-based improved subtractive hybridization is an effective approach to identify differentially expressed genes. Many genes, both known and unknown, might contribute to the existence of drug-resistant phenotype, through increasing or decreasing their expression.