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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。并提示外源性（ＣＡ）１４可为研究环境因素对人体肿瘤ＤＮＡ突变影响提供直接的观察指标
Objective To use RER (replicationerror) phenotype human tumor cell lines to observe the frequent mutations of microsatellite sequences (MS) in tumor cells in vitro to study the genetic instability of human tumor cell genomic DNA. Methods The recombinant plasmid pCMV-CAR containing the artificially synthesized simple repeat sequence (CA) 14 and LacZ marker gene was transfected into RER+ or RER-human colon cancer cell lines by Lipofection method. Insertion of (CA) 14 into the LacZ coding sequence interferes with the correct reading of the LacZ gene. When a base deletion or insertion mutation occurs in the (CA)14 sequence and the number of bases becomes a multiple of 3, the LacZ gene is restored in frame, resulting in the expression of a bioactive β-galactosidase. The latter was detected by X-gal staining. Results The cell clones transfected with pCMV-CAR were selected and cultured by Hygromicin. Some RER+ plasmid transfected clones were observed. LacZ could be read on the correct code. After X-gal staining, the cells turned blue. The blue cells continue to exist during the passage of the established strains. The RER-cell line, pCMV-CAR transfected clones stained with X-gal did not show any blue cells. Conclusion Exogenous (CA) 14 sequences can undergo base loss or insertion mutations in RER+ cell-transfected clones. It directly reflects the DNA instability and complex mutation status of tumor cells. It is suggested that exogenous (CA) 14 can provide direct observational indicators for studying the impact of environmental factors on DNA mutations in human tumors.