特异点突变型p53 minigene对人肺癌PG细胞的转染效应  

Specific point mutate p53 mini gene transfectimg effects on biological behaviors of a human cancer cell line PG derived from human pulmonary giant carcinoma

作　　者：谢建武[1,2,2,3] 方伟岗[1,2,2,3] 惠培[1,2,2,3] 李宝林 李红梅[1,2,2,3] 钟镐镐 郑杰[1,2,2,3] 陈碧芬 吴秉铨[1,2,2,3] 

机构地区：[1]北京医科大学病理学系 [2]福建医科大学分子医学研究室 [3]美国耶鲁大学病理学系

出　　处：《中华医学杂志》1999年第1期57-60,共4页National Medical Journal of China

基　　金：康奈尔中国基金;福建省重点自然科学基金;福建省医药卫生科技项目基金

摘　　要：目的观察一种编码密码子１７２Ａｒｇ→Ｌｅｕ的特异点突变型小鼠ｐ５３ｍｉｎｉｇｅｎｅ对恶性肿瘤细胞的转染效应，并探讨该基因在恶性肿瘤基因治疗中的潜在价值。方法应用ＬｉｐｏｆｅｃｔａＭＩＮＥ和电穿孔法，把该特异点突变型ｐ５３ｍｉｎｉｇｅｎｅ的表达载体与表达载体ＰＣＭＶｎｅｏ共转染ｐ５３错义突变的人肺癌ＰＧ细胞系，并以野生型和相应的密码子１７２Ａｒｇ→Ｈｉｓ点突变型ｐ５３ｍｉｎｉｇｅｎｅ以及空载体转染为对照。应用小鼠特异性ｐ５３基因反转录聚合酶链反应和Ｎｏｒｔｈｅｒｎ杂交方法，检测外源性ｐ５３基因的ｍＲＮＡ转录。观察转基因后Ｇ４１８抗性克隆的克隆形成、细胞生长状态以及对抗癌药的敏感性改变，并应用脱氧核苷酰转移酶原位标记法分析该基因转染对细胞凋亡的影响。结果密码子１７２Ａｒｇ→Ｌｅｕ点突变型小鼠ｐ５３基因组ｍｉｎｉｇｅｎｅ转染后能较强抑制ＰＧ细胞Ｇ４１８抗性克隆的形成，细胞难以传代；基因瞬时表达后能导致细胞凋亡，提高ＰＧ细胞对抗癌药的敏感性，具有较野生型ｐ５３基因更强的抑瘤能力。结论密码子１７２Ａｒｇ→Ｌｅｕ特异点突变型ｐ５３ｍｉｎｉｇｅｎｅ对肿瘤基因治疗有重要的潜在应用价值。Objectives To explore the suppressive effects of a murine genomic p53 minigene containing an Arg→Leu substitution at its encoding amino acid 172 on biological behaviors of human carcinoma cell and evaluate its potential application in cancer gene therapy. Methods By LipofectaMINE and electraporation methods, this mutant p53 gene which lacked of exon 1 and intron 1 expression vector driven by CMV promoter was co transfected with PCMVneo into PG cell in which dominant negative p53 pre exists. A wild type and another kind of genomic mutate type p53 gene expression vector were transfected. The latter p53 gene encoding protein contained an Arg→His substitution at the same position, and pBLuscript plasmid was used as control. All transfectants were screened by 500 μg/ml geneticin and identified by mouse specific p53 mRNA RT PCR and Northern blot analysis. After transfection, the biological behavior changes were studied by colony formation and TUNEL test together with in situ clone regression for chemosensitivity of anti cancer drugs. Results The transfecting effects of this unusual p53 gene were surprisingly strong. They were more significant than those of the wild type p53 and could suppress the formation of transgenic colonies and passage. The transgenic colonies were sensitive to be treated in adromycin and 5 Fu, and the gene transient expression could result in cell apoptosis. Conclusion Codon 172 mutant (Arg→Leu) p53 genomic DNA exhibited a strong suppressive transfecting effects on carcinoma cell, so it is a possible candidate to be used in cancer gene therapy.

关 键 词：肺肿瘤 转染 PG细胞 P53基因 

分 类 号：R734.2[医药卫生—肿瘤]