胞嘧啶脱氨酶尿嘧啶-磷酸核糖基转移酶融合基因/5-氟胞嘧啶治疗鼻咽癌的实验研究  

Experimental study of treating nasopharyngeal carcinoma with fusion cytosine deaminase-uracil phosphoribosyltransferase suicide gene and 5-Flurocytosine

作　　者：唐瑶云[1] 钟宇[1] 赵素萍[1] 肖健云[1] 

机构地区：[1]中南大学湘雅医院耳鼻咽喉科,湖南长沙410008

出　　处：《中国耳鼻咽喉头颈外科》2006年第11期766-770,共5页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：国家自然科学基金(30300414)

摘　　要：目的研究胞嘧啶脱氨酶-尿嘧啶磷酸核糖基转移酶融合基因／5-氟胞嘧啶（fusion cytosine deaminaseuracil phosphoribosyl transferase gene／5-Flurocy-tosine，FCU／5-FC）对鼻咽癌CNE-2细胞的杀伤效应以及对鼻咽癌移植瘤生长的抑制作用。方法构建表达质粒载体pcDNA3．1（-）CMVe·Egr-1·FCU，BamH Ⅰ酶切鉴定重组质粒，分析FCU基因和融合启动子CMVe·Egr-1的序列。电穿孔法将重组质粒转染CNE-2细胞，300μg／ml-600μg／mlG418筛选14天获得稳定表达FCU基因的CNE-2细胞，RT—PCR鉴定FCU基因的表达。以转染空白载体pcDNA3．1（-）的CNE-2细胞为对照，MTT法观察5-FC对表达FCU基因的CNE-2细胞生长的抑制作用，考察FCU基因的细胞旁杀效应。5×10^6的CNE-2细胞接种裸鼠右腋下，建立鼻咽癌裸鼠移植瘤模型，12天后将裸鼠随机分成3组：CU组，CU＋PBS组和CU＋5-FC组，观察不同处理对移植瘤生长的抑制作用。结果酶切和序列分析证明重组质粒含完整的FCU基因和Egr-1启动子的核心序列，RT-PCR从转染细胞总RNA中扩出330bp的预期片段。表达FCU基因的CNE-2细胞在5-FC干预下，生长受到抑制。随着表达FCU基因的细胞比例增加，细胞存活率相应地下降。CU组，CU＋PBS组移植瘤体积与CU＋5-FC组有显著性差异（P〈0．05）。结论表达FCU基因的CNE-2细胞可以被5-FC杀伤，FCU／5-FC系统抑制了移植瘤生长，为治疗鼻咽癌提供了新的策略。OBJECTIVE To study the killing effect of fusion cytosine deaminase-uracil- phosphoribosyltransferase gene /5-Flurocytosine （FCU/5-FC） on the nasopharyngeal carcinoma cell line CNE-2 cells in vitro and the antitumour activity was also observed in situ. METHODS Constructed plasmid pcDNA3.1（-）CMVe.Egr-1.FCU was verified by enzyme digestion of BamH Ⅰ and automatic sequence analysis, then it was introduced into CNE-2 cells by electroperforation to yield cells expressing of FCU stably after selecting with G418（300μg/ml） for 14 days. The expression of FCU mRNA in transfected CNE-2 cells was tested by RT-PCR. Compared with CNE-2 cells transferred with pcDNA3.1（-）, in vitro chemosensitivity of FCU expressing CNE-2 cells to 5-FC was detected by MTT assay. Furthermore, bystander effect of FCU was analyzed by MTT assay. At last NPC model was employed by inoculating CNE-2 cells in the right flank of nude mice and after 12 days of inoculation, those rats were randomly divided into three groups（n=8）： CU group, CU＋PBS group, CU＋5-FC group. The suppression of NPC growth in model was observed after giving different treatments. RESULTS The recombinant plasmid contained full-length coding region sequence of FCU gene and core sequence of Egr-1 promoter. A anticipated 330bp fragment was amplified from total RNA of FCU-expressing CNE-2 cells by RT-PCR. In vitro study, FCU-positive CNE-2 cells were killed by 5-FC while FCU-untransferred CNE-2 cells was killed undistinctively, and the relative survival rates of two groups had significant difference（P〈0.01）, moreover a powerful bystander effect of FCU in killing CNE-2 cells was approved. In situ study, the tumor volume of CU＋5-FC group was different with that of CU group or CU＋PBS group, but no significant difference existed between CU group and CU＋PBS group（P〉0.05）. CONCLUSION FCU-expressing CNE-2 cells can be killed in presence of 5-FC, and the xerografts of NPC were inhibited by FCU/5-FC system, which made of the system a candidate for tre

关 键 词：胞嘧啶脱氨酶 尿嘧啶 糖基转移酶 基因疗法 鼻咽肿瘤 

分 类 号：R739.63[医药卫生—肿瘤]