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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药物罗丹明的蓄积,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克隆耐药相关基因。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
BACKGROUND & OBJECTIVE: Chemotherapy is the most important adjuvant therapy for nasopharyngeal carcinoma. However, the drug resistance of cancer cells often leads to the failure of drug treatment. Therefore, screening of genes related to drug resistance in nasopharyngeal carcinoma and searching for molecules that reverse drug resistance Targets have important practical significance. In this study, nasopharyngeal carcinoma drug-sensitive cell CNE2 was used as the parent cell to induce the establishment of drug-resistant cell lines. On the basis of this, we screened the genes related to drug resistance in nasopharyngeal carcinoma and explored the molecular mechanism of drug resistance. Methods: The human nasopharyngeal carcinoma cell line CNE2 / DDP was induced by cisplatin (DDP) as inducer and the combination of high dose and progressive dose. The drug sensitivity was determined by MTT assay. The cell cycle and the accumulation of intracellular fluorescent drug rhodamine were determined by flow cytometry. The cell growth curve, doubling time and cell morphology were observed. Subsequently, the resistance-related genes were screened and cloned by subtractive hybridization based on PCR technology. Reverse dot blot identification exclusion false positive, DNA sequencing analysis of differentially expressed fragments, RT-PCR differentially expressed gene fragments for further verification. Results: The drug resistance index of human nasopharyngeal carcinoma cell line CNE2 / DDP against DDP was 27.9, which was resistant to 5-fluorouracil (5-FU) and vincristine (VCR) The indices were up to 227.9 and 55.5, respectively, indicating their multidrug resistance profile. Flow cytometry showed that the accumulation of drug-resistant intracellular rhodamine was significantly lower than that of the sensitive cells (12.98 vs. 243.42). Microscopic observation of drug-resistant cells smaller, round shape, cell doubling time was significantly longer (26hvs.19h). Subtraction hybridization found six different tables