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氯化两面针碱(Nitidine chloride,NC)是从芸香科植物两面针根部提取的一种天然生物碱,具有很好的抗肿瘤活性,在体外能抑制鼻咽癌,肺癌等细胞的增殖。研究表明NC可导致细胞周期G2/M期阻滞,并诱导细胞凋亡。E2F/RB在细胞周期调控中起重要作用,它们与CyclinD1、CDK4、CyclinE、CDK2构成复杂的反馈调控网路,调节细胞从G1期到S期的过渡,推动了细胞周期的转变。另外,研究表明E2F是重要的转录因子,参与了基因的转录调控。研究肝癌细胞SMMC-7721经两面针碱处理后RB/E2F通路的相关分子水平变化,以及氯化两面针碱在RB/E2F途径抑制肝癌细胞增殖,参与细胞凋亡作用的机制。应用CCK8方法测定NC对SMMC-7721细胞存活率的影响;Annexin-V和碘化丙碇(propidium i-odide,PI)双染法检测细胞凋亡和坏死情况;RT-PCR检测不同剂量NC对E2F、RB mRNA表达影响;WB检测E2F蛋白含量的变化。结果显示NC可抑制肝癌细胞增殖,降低细胞存活率,促进细胞凋亡,明显降低细胞中E2F、RB mRNA和E2F蛋白的表达,并呈浓度依赖性。NC通过作用于E2F/RB调控通路,抑制了E2F/RB的表达,参与了细胞的增殖抑制和诱导了细胞凋亡。
Nitidine chloride (NC), a natural alkaloid extracted from the roots of Rutaceae plants, has good anti-tumor activity and can inhibit the proliferation of nasopharyngeal carcinoma, lung cancer and other cells in vitro. Studies have shown that NC can lead to cell cycle G2 / M arrest, and induce apoptosis. E2F / RB plays an important role in the regulation of cell cycle. CyclinD1, CDK4, CyclinE and CDK2 play an important role in the regulation of E2F / RB, which regulate the transition from G1 phase to S phase and promote the cell cycle. In addition, studies have shown that E2F is an important transcription factor involved in the transcriptional regulation of genes. To study the changes of molecular level of RB / E2F pathway in hepatoma cell line SMMC-7721 after treating with both sides alkaloids and the mechanism of nitidine chloride on the proliferation of hepatocarcinoma cell line RB / E2F and its role in apoptosis. The effect of NC on the survival rate of SMMC-7721 cells was determined by CCK8 assay; the apoptosis and necrosis of cells were detected by Annexin-V and propidium iodide (PI) staining; E2F, RB mRNA expression; WB detection E2F protein content changes. The results showed that NC could inhibit the proliferation of hepatocellular carcinoma cells, reduce the cell survival rate, promote apoptosis, significantly reduce the expression of E2F, RB mRNA and E2F protein in a concentration-dependent manner. NC through the role of E2F / RB regulatory pathway, inhibiting the expression of E2F / RB, involved in cell proliferation inhibition and induction of apoptosis.