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目的 DNA甲基化模式的改变伴随着永生性细胞系的确立,生长调控基因CpG岛的重新甲基化可能导致了其转录的不活跃,在体外培养的情况下,使得肿瘤细胞选择了有利于生长的条件,我们用DNA甲基转移酶抑制剂5-脱氧杂氮胞苷处理两株肝癌细胞,研究其对肝癌细胞的影响,探讨DNA甲基化异常与肝细胞癌间的相关性及5-脱氧杂氮胞苷对肝癌细胞株恶性生物学行为的影响及其机制。 方法 用5-脱氧杂氮胞苷处理肝癌细胞株SMMC-7721和HePG2,然后采用相差显微镜观察药物处理前后细胞的形态变化,采用MTT法观察细胞的生长速度变化,采用流式细胞仪检测细胞周期、细胞凋亡率、P16蛋白表达的变化,采用RT-PCR法比较p16和甲基转移酶mRNA表达量的变化,比较裸鼠致瘤性的大小。 结果 5-脱氧杂氮胞苷处理后细胞形态趋于规则,生长速度减慢,SMMC-7721细胞,G1期细胞增加了8.5％,而S期和C_2/M朗细胞分别减少了47.8％和10.4％,HePG2细胞,G_1期细胞增加了3.5％,S期减少了46.2％,G_2/M期增加了23.7％,两细胞凋亡率分别增加了91.6％和133.3％,P16蛋白表达分别增加了23.4％和20.9％,p16 mRNA表达增高,而DNA甲基转移酶mRNA表达明显降低,裸鼠皮下移植瘤生长减慢。 结论 肝细胞癌的发生与DNA甲基化异常有关,甲基化酶抑制剂5-脱氧杂氮胞苷可能通过抑制甲?
Changes in target DNA methylation patterns are accompanied by the establishment of immortalized cell lines. The remethylation of the growth regulatory gene CpG island may lead to inactive transcription, and in the case of in vitro culture, the selection of tumor cells favors Under the conditions of growth, we treated two hepatoma cells with the DNA methyltransferase inhibitor 5-deaza cytidine to study their effects on hepatoma cells, and to explore the correlation between DNA methylation abnormalities and hepatocellular carcinoma and 5 Effect of deoxyazepanidine on malignant biological behaviors of hepatocellular carcinoma cell lines and its mechanism . Methods The hepatoma cell line SMMC-7721 and HePG2 were treated with 5-deoxyazacytidine. Morphological changes of the cells before and after drug treatment were observed by phase-contrast microscope. The growth rate of cells was observed by MTT assay, and the cell cycle was detected by flow cytometry. The changes of apoptosis rate and P16 protein expression were compared using RT-PCR method to compare the expression of p16 and methyltransferase mRNA and compare the tumorigenicity of nude mice. Results After treatment with 5-aza-deazacitidine, the morphology of cells tended to be regular and the growth rate was slowed down. SMMC-7721 cells and G1 phase cells increased by 8.5%, while S phase and C_2/M Lang cells decreased by 47.8% and 10.4, respectively. %, HepG2 cells, G1 phase cells increased by 3.5%, S phase decreased by 46.2%, G2 / M phase increased by 23.7%, the two cell apoptosis rate increased by 91.6% and 133.3%, respectively, P16 protein expression increased by 23.4 % and 20.9%, p16 mRNA expression increased, while DNA methyltransferase mRNA expression was significantly reduced, nude mice subcutaneous transplant tumor growth was slowed. Conclusion The occurrence of hepatocellular carcinoma is related to DNA methylation abnormalities. The methylase inhibitor 5-deoxy azacitidine may inhibit A by methylation.