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目的:探讨夜香树3种甾体皂苷(SSCN)对肝癌细胞株BEL-7404增殖抑制作用及其机制。方法:夜香树叶用有机溶剂提取并鉴定3种SSCN,体外培养BEL-7404细胞,采用细胞集落形成法体外测定SSCN对人肝癌细胞株BEL-7404增殖的影响;流式细胞技术检测细胞周期与凋亡率变化;单细胞凝胶电泳(SCGE)分析细胞DNA损伤;用质粒PBR322解旋反应观察SSCN对肿瘤BEL-7404细胞拓扑异构酶Ⅱ的影响。结果:3种SSCN对肝癌细胞BEL7404有明显的抑制作用,并呈剂量-时间依赖性,流式细胞仪分析结果显示,G0/G1和G2/M期细胞数增多,S期减少,呈现细胞阻滞现象,并伴有大量的细胞凋亡。SSCN可以引起DNA损伤,对TopoⅡ活性有抑制作用,可抑制TopoⅡ介导的质粒DNA超螺旋的解旋。结论:SSCN对BEL-7404细胞具有增殖抑制和诱导凋亡作用,其机制可能是:①阻滞细胞于G2/M期,最终导致细胞死亡。②损伤DNA,造成DNA双链断裂。③抑制Topo II的催化活性,使DNA超螺旋增加。
Objective: To investigate the inhibitory effect of three kinds of steroidal saponin (SSCN) on the proliferation of hepatocellular carcinoma cell line BEL-7404 and its mechanism. Methods: Three kinds of SSCN were extracted and identified by organic solvent. BEL-7404 cells were cultured in vitro. The effects of SSCN on the proliferation of human hepatocellular carcinoma cell line BEL-7404 were determined by colony forming assay. The cell cycle and apoptosis were detected by flow cytometry Cell cycle was analyzed by single cell gel electrophoresis (SCGE). The effect of SSCN on topoisomerase Ⅱ in BEL-7404 cells was observed by using the plasmid PBR322. Results: The three kinds of SSCN had a significant inhibitory effect on BEL7404 cells in a dose-and time-dependent manner. Flow cytometry analysis showed that the number of cells increased at G0 / G1 and G2 / M phase and decreased in S phase, showing cell resistance Hysteresis, accompanied by a large number of apoptosis. SSCN can cause DNA damage, inhibit Topo Ⅱ activity and inhibit Topo Ⅱ-mediated unwinding of supercoiled plasmid DNA. CONCLUSIONS: SSCN can inhibit proliferation and induce apoptosis of BEL-7404 cells. Its mechanism may be as follows: ① arrest cells in G2 / M phase and ultimately lead to cell death. ② DNA damage, resulting in DNA double-strand breaks. ③ inhibit the catalytic activity of Topo II, DNA supercoiled increase.