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目的研究左旋棉酚联合低浓度阿霉素(ADM)诱导纤维肉瘤HT1080细胞凋亡及其作用机制。方法采用噻唑蓝(MTT)检测药物对细胞的抗增殖作用,应用瑞氏-姬姆萨(WG)染色、赫斯特33258(hoechst 33258)染色、透射电镜观察细胞的形态学变化,流式细胞仪检测细胞的凋亡率和细胞周期。蛋白免疫印迹法(Western blotting)检测Bcl-2、Bax蛋白的表达,比色分析法检测细胞半胱氨酸天冬氨酸蛋白酶(caspase)-3、caspase-9的活性变化。结果左旋棉酚与低浓度ADM对HT1080细胞具有抑制增殖作用,联合应用作用增强,有浓度与时间依赖性。药物作用后出现细胞凋亡的特征性改变,琼脂糖凝胶电泳观察到梯状带型,流式细胞仪检测发现两药能诱导肿瘤细胞凋亡,联合应用更强。左旋棉酚使细胞阻滞于G1期,ADM使细胞阻滞于S期,联合应用S期的细胞明显增加。细胞内Bcl-2蛋白表达减少,Bax蛋白表达增多,同时出现caspase-9、caspase-3的相继活化,联合应用变化更加明显。结论左旋棉酚与低浓度ADM对纤维肉瘤HT1080细胞有明显抑制增殖及诱导凋亡的作用,联合应用作用加强。
Objective To study the apoptosis of fibroblast sarcoma HT1080 induced by L-Gossypol and low concentration of doxorubicin (ADM) and its mechanism. Methods The antiproliferative effects of the drugs on cells were detected by MTT assay. The cells were stained with Wright-Giemsa (WG) staining, hoechst 33258 staining and transmission electron microscopy. Flow cytometry Instrument detection of cell apoptosis rate and cell cycle. The protein expression of Bcl-2 and Bax was detected by Western blotting. The changes of caspase-3 and caspase-9 activity were detected by colorimetric assay. Results L-gossypol and ADM at a low concentration could inhibit the proliferation of HT1080 cells. The combination of L-gossypol and low concentration of ADM could enhance the effect of combination with time and concentration. Apoptosis was observed after drug treatment, and ladder-like bands were observed by agarose gel electrophoresis. Flow cytometry showed that the two drugs could induce apoptosis of tumor cells, and the combined application was stronger. L-Gossypol blocked cells in G1 phase, ADM blocked cells in S phase, and cells in combination with S phase increased significantly. Bcl-2 protein expression decreased, Bax protein expression increased, while the caspase-9, caspase-3 have been activated, the joint application of more obvious changes. Conclusion L-gossypol and low-concentration ADM can significantly inhibit the proliferation and induce apoptosis of fibrosarcoma HT1080 cells, and the combined effect is stronger.