人鼻咽癌順鉑耐藥細胞系(CNE2/DDP)的建立及耐藥相關基因的篩選

背景與目的:化療是鼻咽癌最主要的輔助治療手段,然而癌細胞耐藥性的產生常常導致藥物治療的失敗,因此,篩查鼻咽癌耐藥相關基因、尋找逆轉藥物耐受的分子靶點具有重要的現實意義。本研究首先用鼻咽癌藥物敏感細胞CNE2作為親本細胞誘導建立耐藥細胞系,并在此基礎上,篩選鼻咽癌耐藥相關基因,探討耐藥性產生的分子機制。方法:以順鉑(cisplatin,DDP)為誘導劑,采用大劑量沖擊與劑量逐漸遞加相結合的方法,誘導建立人鼻咽癌耐藥細胞系CNE2/DDP。采用MTT法測定藥物的敏感性、流式細胞術測定細胞周期及細胞內熒光藥物羅丹明的蓄積,并進行細胞生長曲線測繪、倍增時間測定及細胞形態學觀察。隨后,采用基于PCR技術改良的消減雜交法篩選并克隆耐藥相關基因。反向點雜交鑒定排除假陽性,DNA測序分析差異表達片段,RT-PCR對差異表達的基因片段做進一步驗證。結果:所建立的人鼻咽癌耐藥細胞系CNE2/DDP對DDP的耐藥指數為27.9,對5-氟尿嘧啶(5-fluorouracil,5-FU)及長春新堿(vincristine,VCR)的耐藥指數分別可達227.9和55.5,表明其具有多藥耐藥的特征。流式細胞測定顯示耐藥細胞內羅丹明的蓄積明顯低于敏感細胞(12.98vs.243.62)。鏡下觀察耐藥細胞體積變小,形態變圓,細胞倍增時間明顯延長(26hvs.19h)。消減雜交發現了6個差異表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.link_to_subscribed_fulltex