HDAC抑制剂SAHA对人肝癌细胞分化诱导作用的研究

目的 探讨组蛋白去乙酰化酶(histone deacetylases,HDAC)抑制剂SAHA(suberoylanilide hydroxamic acid)对人肝癌SMMC-7721细胞的分化诱导作用.方法 倒置显微镜观察SAHA对SMMC-7721细胞形态的影响;MTT比色法测定SAHA对SMMC-7721细胞增殖的抑制情况;免疫细胞化学检测SAHA对SMMC-7721细胞中甲胎蛋白(AFP)和增殖细胞核抗原(PCNA)表达的影响;流式细胞术(FCM)分析细胞周期;RT-PCR方法榆测处理前后SMMC-7721细胞p21WAF1基因mRNA的表达变化.结果 实验组细胞增殖速度显著减慢,与正常细胞形念变化相似;MTT比色法测定结果显示不同浓度SAHA对SMMC-7721细胞的增殖均有抑制作用,并有明显的剂量依赖和时间依赖关系;免疫细胞化学检测显示SAHA能显著降低PCNA和AFP在SMMC-7721细胞中的表达;流式细胞仪检测结果显示,SMMC-7721细胞经SAHA处理后,G0/G1期细胞明显增加,S期细胞则明显减少,细胞被阻滞于G0/G1期;RT-PCR检测结果表明,实验组细胞中p21WAF1 mRNA的表达明显增加.结论 SAHA对人肝癌细胞具有显著的诱导分化作用,诱导肝癌细胞分化的机理可能与抑制HDAC的活性,上调p21 WAF1 mRNA表达,及阻滞肝癌细胞G0/G1期有关.

Effects of SAHA on the differentiation of HCC SMMC-7721 cells

Objective To investigate the effects of SAHA on the differentiation of human hepatocellular carcinoma(HCC)SMMC-7721 cells.Methods Cell morphology was examined by light microscopy;Cell viability was determined by MTT assay;The expression of Alpha-fetoprotein(AFP)and proliferating cell nuclear antigen(PCNA)was analyzed with immunocytochemistry;Flow cytometry(FCM)was used to investigate the cell cycle;The expression of p21 WAF1 mRNA was detected by semi-quantitative RT-PCR.Results Light microscopy showed that SMMC-7721 cells induced by SAHA underwent restorational alterations in morphology which were different from those of nontreated cells but were similar to those of normal cells;MTT assay showed that SAHA inhibited the proliferation of SMMC-7721 cells in a dose-dependent and time-dependent way;Immunocytochemistry assay showed that the expression of AFP and PCNA decreased significantly;FCM analysis showed SAHA could arrest SMMC-7721 cells at G0/G1 phase,with an accumulation of the cells at G0/G1 phase while a decrease of cells at S phase;Semi-quantitative RT-PCR detection revealed that the expression of p21 WAF1mRNA was upregulated remarkably in the cells treated with SAHA.Conclusion SAHA could induce differentiation of human HCC SMMC-7721 cells inhibiting enzymatic activity of HDAC,upregulating the expression of p21 WAFlmRNA as well as causing arrest of HCC cells at G0/G1 phase.