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目的:研究雷公藤内酯醇(triptolide,TP)对人肝癌SMMC-7721细胞增殖的影响以及对P53基因的去甲基化作用。方法:MTT法检测TP对SMMC-7721细胞增殖的影响,甲基特异性PCR检测TP对SMMC-7721细胞P53基因甲基化的影响,RT-PCR检测SMMC-7721细胞甲基转移酶DNMT1、DNMT3a、DNMT3bmRNA的表达,Western blotting检测SMMC-7721细胞中P53蛋白的表达。结果:TP剂量依赖性抑制SMMC-7721细胞的增殖(P<0.05),40 ng/ml时的抑制率达(73.5±3.02)%,其半数抑制浓度(IC50)约为20 ng/ml。TP显著抑制SMMC-7721细胞中DNMT1、DNMT3a、DNMT3bmRNA的表达(P<0.05,P<0.01);TP作用后P53基因的高甲基化被逆转,并呈剂量依赖性;TP可显著增强SMMC-7721细胞中P53蛋白的表达。结论:TP可通过抑制甲基转移酶使P53基因去甲基化,促进P53蛋白的表达,从而抑制SMMC-7721细胞的增殖。
Objective: To study the effects of triptolide (TP) on the proliferation of human hepatocellular carcinoma SMMC-7721 cells and the demethylation of P53 gene. Methods: MTT assay was used to detect the effect of TP on the proliferation of SMMC-7721 cells. Methyl-specific PCR was used to detect the effect of TP on the methylation of P53 gene in SMMC-7721 cells. The expressions of DNMT1, DNMT3a , DNMT3bmRNA expression, Western blotting detection of P53 protein expression in SMMC-7721 cells. Results: TP dose-dependently inhibited the proliferation of SMMC-7721 cells (P <0.05). The inhibitory rate of TP at 40 ng / ml was (73.5 ± 3.02)% and the IC50 was about 20 ng / ml. TP significantly inhibited the expression of DNMT1, DNMT3a and DNMT3b mRNA in SMMC-7721 cells (P <0.05, P <0.01); TP increased the hypermethylation of P53 gene in a dose-dependent manner; TP increased the expression of DNMT1, In P53 protein expression. Conclusion: TP can demethylate P53 gene by inhibiting methyltransferase and promote the expression of P53 protein, thereby inhibiting the proliferation of SMMC-7721 cells.