外源性微卫星序列转染RER^+细胞株观察肿瘤细胞遗传不稳定性的研究  

INVESTIGATION OF THE GENETIC INSTABILITY OF TUMOR CELLS BY TRANSFECTION OF RER+ CELL LINES WITH EXOGENOUS MICROSATELLITE SEQUENCE

作　　者：汪承亚[1] 丁小键[1] 倪黎[1] 盛瑞兰[1] 王兰华[1] 

机构地区：[1]南京医科大学第一附属医院中心实验室

出　　处：《中华医学遗传学杂志》1998年第4期246-249,共4页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金

摘　　要：目的利用ＲＥＲ（ｒｅｐｌｉｃａｔｉｏｎｅｒｒｏｒ）表型的人肿瘤细胞株，在体外观察肿瘤细胞微卫星序列（ＭＳ）频发突变，研究人类肿瘤细胞基因组ＤＮＡ的遗传不稳定性。方法把含有人工合成的简单重复序列（ＣＡ）１４和ＬａｃＺ标志基因的重组质粒ｐＣＭＶ－ＣＡＲ经Ｌｉｐｏｆｅｃｔｉｏｎ方法转染ＲＥＲ＋或ＲＥＲ－人结肠癌细胞株。（ＣＡ）１４插入ＬａｃＺ编码序列之中，干扰了ＬａｃＺ基因的正确读码。当（ＣＡ）１４序列发生碱基缺失或插入突变，碱基数变为３的倍数时ＬａｃＺ基因读码框恢复，表达产生有生物活性的β－半乳糖苷酶。后者通过Ｘ－ｇａｌ染色检出。结果转染ｐＣＭＶ－ＣＡＲ的细胞克隆经Ｈｙｇｒｏｍｉｃｉｎ筛选并建株培养，观察到部份ＲＥＲ＋的质粒转染克隆，ＬａｃＺ能正确读码表达，经Ｘ－ｇａｌ染色试验，细胞变蓝。蓝细胞在建株培养后的传代过程中持续存在。而ＲＥＲ－细胞株，ｐＣＭＶ－ＣＡＲ转染克隆用Ｘ－ｇａｌ染色未见任何蓝色细胞。结论外源性（ＣＡ）１４序列在ＲＥＲ＋细胞转染克隆中可以发生碱基丢失或插入突变。直接反映了肿瘤细胞ＤＮＡ的不稳定性和复杂的突变状态。Objective Frequent alterations of microsatellite sequence of cancer cells were found recently in a substantial fraction of human cancers including hereditary nonpolyposis colon cancer. This paper aimed to investigate the genetic instability of tumor cells by using microsatellite instability(MI) as the marker in vitro.Methods Two RER+ cell lines(replication error phenotype), RKO and HCT116 and one RER- cell line, sw480, were used as the hosts for transfection with an episomal plasmid, pCMVCAR, containing an exogenous(CA)14 repeat which was inserted within the coding sequence of lacZ reporter gene and thus made lacZ misreading. The transfectant clones were selected and established by hygromicin. Expression and production of lacZ reporter gene of restored reading frame were detected with Xgal staining assay.Results After hygromicin selection, stable pCMVCAR transfectant clones were established. It was shown that mutation of deletion or insertion within (CA)14 occured in the transfectant RER+ cells but not in the RERcells. The mutation restored normal reading frame of lacZ gene, and resulted in expression and production of bioactive beta galactosidase which was detected with Xgal staining. This feature of the transfectant clones was maintained during culture passages.Conclusion The alterations of the exogenous (CA)14 repeat in the transfectant RER+ clones revealed genetic instability and complicated mutation status of cancer cells. It is suggested that the exogenous (CA)14 in transfectant clones could be a useful target sequence for monitoring the effects of environmental agents on MI of human cancer cells.

关 键 词：微卫星序列 穿梭质粒 肿瘤细胞 遗传不稳定性 

分 类 号：R730.4[医药卫生—肿瘤]