化学致癌物DNP致人胚鼻咽上皮细胞转化相关基因的鉴定  

Identification of the Gene Involved in DNP-induced Neoplastic Transformation of Human Embryonic Nasopharyngeal Epithelial Cells

作　　者：段朝军[1] 关勇军[2] 何春梅[2] 李峰[2] 肖志强[1] 陈主初[1] 

机构地区：[1]中南大学湘雅医院医学实验中心,中国湖南长沙410078 [2]中南大学肿瘤研究所,中国湖南长沙410078

出　　处：《生命科学研究》2005年第1期84-89,共6页Life Science Research

基　　金：国家自然科学基金资助项目(39470777);国家自然科学基金资助项目(3977064)

摘　　要：为了探讨DNP致癌的分子机理,鉴定出化学致癌物二亚硝基哌嗪(DNP)致人胚鼻咽上皮细胞转化相关的基因及其活化方式.采用DNA共转染、裸鼠致瘤性试验、Southern杂交、PCR测序和序列同源性比较分析等,对DNP转化的人胚鼻咽上皮细胞株HENE-DNP进行研究.经过两轮DNA共转染和裸鼠致瘤性实验,Southern杂交表明,裸鼠肿瘤DNA中均含有人特异性高度重复序列Alu.用人Ha-ras、Ki-ras及N-ras癌基因特异性引物对裸鼠肿瘤DNA进行PCR扩增,仅能扩增出人Ha-ras基因相应的片段.Southern杂交进一步证实,裸鼠肿瘤DNA中存在与人Ha-ras基因片段大小一致的杂交带.RT-PCR产物测序,并将测序结果与GenBank进行序列同源性比较分析,发现裸鼠肿瘤中人Ha-ras基因cDNA第26位密码子第2位碱基发生了T→C的转换,编码的氨基酸由亮氨酸相应地变换成丝氨酸.化学致癌物DNP致人胚鼻咽上皮细胞转化相关的基因是Ha-ras,原癌基因c-Ha-ras激活可能是DNP转化人胚鼻咽上皮细胞的分子机制之一.To identify the genes and explore th e molecular mechanism involved in neoplastic transformation of human embryonic nasopharyngeal epi thelial(HENE)cells induced by N,N'-Dinitrosopip erazine(DNP).DNA co-transfection,nude mice tumorig enicity assay,Southern hybridization,polymerase chain reaction(PCR)and DNA sequence analysis were employed to study HENE-DNP,a human embryonic nasopharygneal epithelial cell line transformed by DNP.After co-tr ansfection with genomic DNA of HENE-DNP cells and pKJ1-neo plasmid DNA,the G418-resistant clones of NI H3T3cells were selected and inocula ted into nude mice.Four weeks later tumors occurred in nude mice,and gen omic DNAs were extracted from the tum ors in nude mice.The results from Southern hybridization showed that Alu,a human-origin highly repeated sequence,was present in tumors and concentrated after twice DNA co-transfection and nude mice tumorigenicity assay.When PCR was per-formed with a set of primers designed for human Ha-ras,Ki-ras or N-ras oncogene specific sequence,only an amplicon corresponding to human Ha-ras oncogene primers were found from the tumors in nude mice.Furthermore,it was confirmed by Southern blotting.Using RT-PCR,sequence analysis and homogeneity comparison between the sequence of PCR product and of GenBank database,i t was also demonstrated that the existence of a T →C mutation in codon 26of Ha-ras oncogene amplified from the DNA of tumors i n nude mice,which result in the amino acid sequen ce of RAS protein changed from leucin e to serine.The gene involved in chemical carcinogen DNP-induced tr ansformation is cellular oncogene c -Ha-ras,and its activation may be on e of the molecular mechanisms of DNP-induced neoplastic transformation of human embryonic nasopharyngeal cells.

关 键 词：二亚硝基哌嗪 共转染 转化基因或癌基因 

分 类 号：R392[医药卫生—免疫学]