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目的 以复制错误 (replication error,RER+)表型的人结肠癌细胞株 RKO作为靶细胞 ,研究全反式维甲酸 (all- trans- retinoic acid,ATRA)和茶多酚 (polyphenon,PP)对肿瘤细胞微卫星序列(microsatellite sequence,MS)突变的影响 ,并观察肿瘤细胞内碱基错误配对修复 (mismatch repair,MMR)基因 h ML H1 和 h MSH2 的表达情况。方法 将含有外源性 MS(CA) 1 4 的穿梭质粒 p CMV- CAR转染RER+人结肠癌细胞株 RKO。外源性的 (CA) 1 4 的突变可使质粒标记基因 L ac Z恢复正常读码 ,表达产生β-半乳糖苷酶 ,后者使 X- gal变蓝。全反式维甲酸和茶多酚对 MS的影响可直接通过 X- gal染色结果判断。利用逆转录 -聚合酶链反应方法 ,检测经全反式维甲酸和茶多酚处理的 RKO细胞中碱基错误配对修复基因h ML H1 和 h MSH2 的表达情况。结果 全反式维甲酸 1μmol/ L、0 .1μmol/ L ,茶多酚 3μg/ ml,对 RKO细胞的增殖无明显的影响。作用 1周后 ,均显示对外源性 (CA) 1 4 的突变有明显的抑制效应。但不能诱导h ML H1 和 h MSH2 表达。结论 全反式维甲酸和茶多酚能抑制 RER+细胞中外源性 (CA) 1 4 重复序列突变 ,提示两者对人癌细胞 MS遗传不稳定有保护作用。其作用机理可能不是通过影响 h ML H1 和 h MSH2 的表达而起作?
Objective To investigate the effect of all-trans retinoic acid (ATRA) and polyphenon (PP) on human colon cancer cell line RKO with replication error (RER +) phenotype. (MMR) gene h ML H1 and h MSH2 in tumor cells were observed. Methods The shuttle plasmid pCMV-CAR containing exogenous MS (CA) 1 4 was transfected into RER + human colon cancer cell line RKO. The exogenous (CA) 14 mutation results in the normalization of the plasmid marker gene L ac Z, which produces beta-galactosidase, which turns X-gal blue. The effect of all-trans retinoic acid and tea polyphenols on MS can be directly assessed by X-gal staining. The expression of mismatch repair genes h ML H1 and h MSH2 in RKO cells treated with all-trans retinoic acid and tea polyphenols were detected by reverse transcription polymerase chain reaction (RT-PCR). Results All-trans retinoic acid 1μmol / L, 0.1μmol / L and tea polyphenol 3μg / ml had no significant effect on the proliferation of RKO cells. After 1 week of treatment, all showed a significant inhibitory effect on the mutation of exogenous (CA) 1 4. But failed to induce hMLH1 and hMSH2 expression. Conclusion All-trans retinoic acid and tea polyphenols can inhibit exogenous (CA) 1 4 repeat mutations in RER + cells, suggesting that both of them may have a protective effect on the genetic instability of human cancer cells MS. Its mechanism of action may not be affected by the impact of hMLH1 and hMSH2 expression?