红景天甙体外诱导人肝癌QGY-7703细胞凋亡研究

Study on cell apoptosis of human hepatoma QGY-7703 cell induced by salidroside in vitro culture

【目的】探讨红景天甙(Salidroside)体外诱导人肝癌QGY-7703细胞凋亡的作用及其可能的机制。【方法】以体外培养的人肝癌QGY-7703细胞作为研究对象,采用四甲基偶氮唑盐(MTT)比色法检测红景天甙对QGY-7703细胞生长的抑制作用,采用吖啶橙/溴化乙啶(AO/EB)荧光双染、琼脂糖凝胶电泳及流式细胞术,分析红景天甙对QGY-7703细胞凋亡的诱导作用;采用Western blot免疫印迹法探讨红景天甙诱导QGY-7703细胞凋亡的可能机制。【结果】MTT比色法试验结果表明,0.01,0.1,1,10和100μg/mL红景天甙对QGY-7703细胞生长均有不同程度的抑制作用,细胞生长抑制率分别为13.2%,21.9%,31.4%,46.8%和60.9%,半数抑制质量浓度(IC50)为18.56μg/mL;用10μg/mL红景天甙处理QGY-7703细胞48 h,AO/EB荧光双染后,可观察到个别QGY-7703细胞呈现亮绿色或橘红色,表明细胞发生了凋亡;琼脂糖凝胶电泳结果显示,10μg/mL红景天甙作用QGY-7703细胞24,48和72 h均可导致细胞核内DNA产生有序断裂,形成DNA梯形带;流式细胞术试验结果表明,10μg/mL红景天甙的加入量为100和200μL时,QGY-7703细胞凋亡数分别为178和331个,相应的凋亡率分别为1.8%和3.5%,阴性对照组(10μg/mL红景天甙加入量为0μL)凋亡细胞数为105个,凋亡率仅为1.1%;Western blot免疫印迹分析结果表明,红景天甙下调了Bcl-2和PCNA蛋白的表达量,而使凋亡活化基因Bax与肿瘤抑制基因p53的蛋白表达量增高。【结论】红景天甙对体外培养的QGY-7703细胞具有明显的生长抑制作用和诱导凋亡作用,其诱导凋亡作用与Bcl-2家族成员、p53、Bax和PCNA基因调控有关。

【Objective】 The experiment was conducted to study cell apoptosis and possible mechanism of human hepatoma QGY-7703 cells induced by salidroside in vitro culture.【Method】 With human hepatoma QGY-7703 cells as study object in vitro culture,the QGY-7703 growth inhibition effect of salidroside was measured by methyl thiazolyl tetrazolium（MTT） method;The apoptosis effect of QGY-7703 was analyzed by AO/EB double fluorescent dye staining,agarose gel electrophoresis and flow cells analysis;The possible mechanism of QGY-7703 apoptosis induced by salidroside was studied by western blot.【Result】 The result of MTT method showed that salidroside of 0.01,0.1,1,10 and 100 μg/mL all inhibited the growth of QGY-7703 cells.The cell growth inhibition rate of QGY-7703 was 13.2%,21.9%,31.4%,46.8% and 60.9% respectively and the 50% inhibiting concentration（IC50） was 18.56 μg/mL.By AO/EB double fluorescent dye staining treated with 10 μg/mL salidroside for 48 h,individual cells presented bright green and jacinth.It showed that these cells apoptosised.The DNA in nucleus appeared regular rupture and formed DNA ladder treated with 10 μg/mL salidroside for 24,48,and 72 h in agarose gel electrophoresis experiment.The result of flow cells analysis showed that the apoptosis cells were 178 and 331 and the apoptosis rate was 1.8% and 3.5% respectively when added amount of 10 μg/mL salidroside was 100 μL and 200 μL,but the apoptosis cells were 105 and the apoptosis rate was only 1.1% in the control group（the volume of 10 μg/mL salidroside was 0 μL）.Western blot showed that salidroside down-regulated the expression of Bcl-2 and PCNA proteins,but protein expressions of apoptosis activation gene Bax and tumor suppressor gene p53 were up-regulated.【Conclusion】 The salidroside could inhibit cell growth and induce cell apoptosis of OGY-7703 cells in vitro culture.The effect of apoptosis was related to the gene modulation of Bcl-2 family,p53,Bax and PCNA.